INTRODUCTION:
Glycation is a spontaneous non-enzymatic amino-carbonyl
reaction between sugars and long-lived proteins, lipids and
nucleic acids and it is also one of the post translational
modification processes between the reducing sugars and free
amino groups of proteins (Monnier and Carami., 1981). In the
early stage of glycation the synthesis of intermediates lead to
the formation of Amadori compounds and in late stage
advanced glycation end products (AGEs) are irreversibly
formed as a result of a complex cascade of reactions. Among
the amino groups lysine is a preferred group for non-enzymatic
sugar attachment and subsequent formation of AGEs (Arai et
al., 1987).
The process of glycation occurs continuously in the
body even at normal glucose levels and the damage caused by
the Amadori and AGE products keep accumulating slowly
overtime. The deleterious effects of these products are more
significantly observed when the blood glucose level increases
above the normal level in conditions like hyperglycemia.
Glycation has been linked to various diseases such as diabetes,
cataract, Alzheimer's, dialysis related amyloidosis (DRA),
atherosclerosis, Parkinson's as well as physiological aging
(Thorpe and Bayens, 1996).
The complex process of glycation alters the biological
activities of proteins, their metabolic processes and inhibits the
specific functions of proteins through cross linking,
aggregation, precipitation and produce reactive oxygen species
(ROS) (Suji and Sivakami, 2004). Protein cross-linkage results
in protein aggregates, which then form intracellular proteaseresistant and ubiquitin-proteasome-resistant deposits and
inhibits the intracellular transport of materials. A large amount
of ROS is produced as a result of subsequent modifications of
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from damage.
The conformational modification of plasmid DNA
due to in vitro incubation withseveral sugars or their sugar
phosphates was investigated. Ribose, ribose-5-phosphate
andfructose-6-phosphate damaged the DNA in the presence of
Fe3+ and formed the opencircular as well as linearized the
supercoiled DNA. The extent of damage was differentfor
different sugars and sugar phosphates. It could be due to the
differences in thereactivities of sugars and their phosphates
and also their potential in generating thesuperoxide radicals.
The results showed that the sugar phosphates are highly
reactive and more damaging than sugars. Sugar metabolite
such as MG damaged the DNA faster than sugars. This is
probably due to MG consists two carbonyl groups which
makes it highlyreactive and more damaging than sugars
(Thornalleyet al., 1999).
PLP and P were able to protect DNA damage while
PM lacked DNA protectionability. Those vitamins having high
hydrogen donating ability, show effective antioxidantnature
(Suji and Sivakami, 2007). The weak hydrogen donating
ability of PM makes it amarginally effective antioxidant. The
electron donating para substituents have a stronginfluence on
the radical trapping ability of PM, as they lower the phenolic
O-H bondingdissociation enthalpies (Delange and Glazer,
1989). PLP showed higher hydrogenperoxide radical trapping
capacity. These results indicate that antioxidant property
ofvitamins is probably playing important role in preventing the
damage to DNA.
The effect of various antioxidants was checked on the
DNA damage by addingthese scavengers before (pre) and after
(post) incubating the DNA in the presence of Lys, MG and Fe.
From this study it was seen that NaN3 prevented the DNA
damage bothunder pre and also at post incubation along with
FeCl3. Whereas PLP protected the DNAdamage significantly
only when it was incubated before the addition of FeCl3but it
didnot show any protective effect when added to the damaged
DNA. Pyridoxine prevented the DNA damage partially when
added before the 2 hour incubation with FeCl3and showed no
effect when added after 2 hour incubation with FeCl3. On the
other hand PMwas not able to protect the DNA against the
damaging agents in both pre and postincubation conditions.
Curcumin (Sajithlalet al., 1998) and bhasma
(Pattanaiket al., 2000) have beenknown for their antioxidant
properties and as potent free radical scavengers. In this study,
curcumin and jasadbhasma prevented the DNA damage from
free radicals. Bhasmainhibits DNA damage more effectively
than curcumin. Jasadbhasma inhibited the production of
hydrogen peroxide (43%) by metalcatalyzedglycation system.
On the contrary Curcumin caused enhanced the
hydroxylradical formation (almost 2 fold).
The results presented in this study shows that glucose
and MG can glycate BSAdue to formation of AGEs. Among
these the MG is more reactive in generating browningand in
damaging of BSA than glucose. Curcumin and jasadbhasma
show antiglycatingproperty and prevented the aggregation or
precipitation of the protein.
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Figure 4: pBR 322 DNA (0.5 g) was incubated with lysine (20
mM), MG (20 mM), FeCl3 (100 M) and different
concentrations of various vitamins (5 mM and 10 mM) at 37 C
for 3 hours.
1
10
Figure 5: pBR 322 DNA (0.5 g) was incubated with lysine (20
mM), MG (20 mM), FeCl3 (100 M) (Lanes 2 to 6) at 37 C for
2 hours each, then added antioxidants and further incubated at
37 C for 3 hrs. In lanes 7 to 10 first pBR 322 DNA (0.5 g) was
incubated with lysine (20 mM), MG (20 mM), antioxidants at
37 C for 2 hours, then added FeCl3 and further incubated at 37
C for 3 hrs.
Figure 6: pBR 322 DNA (0.5 g) was incubated with lysine (20
mM), MG (20 mM), FeCl3 (100 M), curcumin (200 g) and
bhasma (200 g) at 37 C for 3 hours.
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REFERENCES :
Ahmad, S., Moinuddin. Dixit, K., Shahab, U., Alam, K., Ali, A. 2011.
Genotoxicity and immunogenicity of DNA-advanced glycation end
products
formed by methylglyoxal and lysine in presence of
2+
Cu .Biochem Biophys Res Commun.407:568-574.
Ames, T. W. 1983. Dietary carcinogens and anticarcinogens. Oxygen
radicals degenerative diseases. Science. 221: 12546-12564.
Arai, K., Maguchi, S., Fujii, S., Ishibashi, H., Oikawa, K., Taniguchi,
N. 1987. Glycation and inactivation of human Cu-Zn-superoxide
dismutase. Identification of the in vitroglycated sites. J Biochem.
262: 16969-16972.
Delange, R. J., Glazer, A. N. 1989. Phycoerythrin fluorescence-based
assay for peroxy radicals: a screen for biologically relevant
protective agents. Anal Biochem. 177: 300-306.
Jakus, V. 2000. The role of free radicals, oxidative stress and
antioxidant systems in diabetic vascular disease. Bratisl Lek
Listy.101: 541-551.
CONCLUSION:
The effect of glycation is more prominent when there is an
accumulation ofsugars and sugars metabolites in the cells and
tissues. These molecules differ in theirpotential in causing
damage to DNA and proteins. In the present study it can be seen
that sugar phosphates cause more extensive damage to DNA
than their corresponding sugars.It can be mainly because of
their reactivities and potential in production of free
radicals.Fructose-6-phosphate is the most effective in
disturbing the supercoiled structure ofplasmid DNA. This can
be due to the amount of hydrogen peroxide produced which
was more for this sugar phosphate as compared to other sugars.
Antioxidants and natural products varied in their
efficacy in preventing the damage to DNA and proteins. There
can be several reasons for this behaviour. These compounds
differ from each other with respect to their capability and
specificity of scavenging the free radicals and at different
concentrations. Among the compounds tested pyridoxamine
showed least preventive activity. This could be due to its low
hydrogen donating capacity (Delange and Glazer, 1989; Suji
and Sivakami, 2007).
It can be concluded from the results presented in this
study that damage to DNA is free radicals mediated and in case
of protein a mechanism based on the accumulation of AGEs
seems to play a critical role in damaging the structures. There
are several other reports in the literature which suggests a
similar mechanism for the structural alteration of biomolecules
(Kikuchiet al.,2003). It can be also suggested from the results
presented in this study that naturalcompounds are very
important for preventing the damage to DNA due to
productaccumulation of early and advanced reactions of
glycation. However, further studies are required to characterize
the exact mechanism of the action of the sugars and
theirmetabolites.
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