ISSN 10227954, Russian Journal of Genetics, 2013, Vol. 49, No. 7, pp. 765–770. © Pleiades Publishing, Inc., 2013.
Original Russian Text © S.A. Smirnikhina, E.S. Voronina, V.V. Strelnikov, A.S. Tanas, A.V. Lavrov, 2013, published in Genetika, 2013, Vol. 49, No. 7, pp. 877–883.
Toxicogenomics is a branch of genetics that studies
the complex genome response to exogenous sub
stances action, including environmental factors, tox
ins, drugs, and chemical substances. Toxicogenomics
is inseparably related to functional genomics and con
siders the effect of toxins on the gene expression,
structure and function, gene–gene interactions, bio
logical pathways, and cellular physiology [1, 2]. DNA
methylation is one of the key mechanisms of variations
in gene expression and genome stability. Methylation
was shown to be associated with a majority of heredi
tary diseases, including cancer [3, 4].
The present study focused on investigating the role
of two mutagens of different mechanisms of action,
dioxidine and methyl methanesulphonate, in varia
tions in the levels of global DNA methylation in
human peripheral whitebloodcell cultures.
MATERIALS AND METHODS
The present study involved 14 almost healthy indi
viduals (five males and nine females) aged 20–40.
Written informed consent was obtained from each
individual. Volunteers had no Xray analysis in the last
six months, no viral infections in the last 3 months,
and had no contact with hazardous industrialchemi
Peripheral blood samples were taken after fasting in
standard heparincontaining tubes (Vacutest, Italy).
The blood was cultivated in RPMI1640 media
(PanEco, Russia) with an addition of 15% bovine serum
albumin (PAA Laboratories, Austria) and phytohemag
glutinin (PHA, PanEco, Russia) (0.01 mg/mL). The
total volume comprised 10 mL with 10% blood.
Dioxidine and methyl methanesulphonate (MMS)
differing in the mechanism of mutagenic action were
used as mutagens. Dioxidine action is based on its pos
sibility to induce the formation of active oxygen forms
. MMS is an alkylating agent of direct action mod
ifying guanine to 7methylguanine and adenine to
3methyladenine, which results in impaired homology
and the inhibition of replication, respectively .
Both mutagens were added after 24 h of culturing in
final concentrations of 0.01 and 0.1 mg/mL (dioxi
dine) and 0.0025 and 0.01 mg/mL (MMS). An analy
sis of cell cultures was also conducted before culturing
and after 25 h of culturing under mutagenfree condi
tions. The duration of culturing was 25 h. The optimal
periods of addition and concentrations have been
detected previously using chromosomal aberrations
method. Selected dioxidine and MMS concentrations
resulted in an increase in aberrations that differed sig
nificantly from the control level; however, no toxic
effect was observed [7–9].
The global methylation was assessed via meth
ylsensitive Comet assay. This method is based on the
addition of two isoschizomers of restriction endonu
that cleave the same restric
tion site, i.e., 5'CCGG3';
is known to cleave
it in the presence of methylation, while
Mutagen Influence with Different Mechanisms of Action on DNA
Global Methylation in Human WholeBlood Lymphocytes in vitro
S. A. Smirnikhina, E. S. Voronina, V. V. Strelnikov, A. S. Tanas, and A. V. Lavrov
Research Center for Medical Genetics, Russian Academy of Medical Sciences, Moscow, 115478 Russia
Received September 20, 2012
—Data that support the evidence of mutagens known to cause epigenetic abnormalities that could
potentially result in genomic instability and the development of cancer rather than to modifications in the
human genome at the gene and chromosomal levels only. The level of global methylation in human lympho
cytes in vitro caused by exposure to two mutagens with different mechanisms of action, i.e., dioxidine and
methyl methanesulphonate (MMS), was demonstrated in the present study. Global methylation was assessed
by methylsensitive comet assay. An increase in the level of global methylation to 45.64% was revealed during
culturing with dioxidine in a concentration of 0.01 mg/mL (
0.001), while the addition of dioxidine in a
concentration of 0.1 mg/mL resulted in a decreased level of methylation up to 42.31% (
0.001). The addi
tion of MMS in concentrations of 0.0025 and 0.01 mg/mL resulted in minor but significant modifications
< 0.05) of the global methylation level ranged within natural variations in global methylation. Accordingly,
the addition of dioxidine in the concentration of 0.1 mg/mL might cause genomic instability and might be
considered a potential carcinogen.