Med Chem Res (2013) 22:674–680
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frequency of cells in first (M1), second (M2) and third
(M3) cell division, and colcemid to accumulate cells in
metaphase, were also purchased from Sigma-Aldrich.
for 20 min with Hoechst 33258 (5 mg/ml), mounted in
saline sodium citrate buffer (SSC) 29 powder (Anidra
Company), and exposed to ‘‘black light’’ for 20 min at
50 °C. Finally, cells were stained with Giemsa and air-
dried for evaluation of the frequency of cells in their first
(M1), second (M2) and third (M3) cell division. Cell
division kinetics was determined by the PRI according to
the formula (Rojas et al., 1992):
Test systems and culture conditions
The karyotype, generation time, plating efficiency, and
absence of mycoplasmal contamination were checked at
regular intervals. Permanent stocks of CHO cells were
stored in liquid nitrogen (-170 °C) and subcultures are
prepared from these stocks for experimental use. At the end
of assay the cells were discarded and a new ampoule
(containing cells from the stock under liquid nitrogen) was
used. Cultures of the cells are grown in F-10 minimal
medium supplemented with 10 % foetal bovine serum,
2 mM L-glutamine and antibiotics (1 % w/v penicillin and
86 mM streptomycin). All incubations were at 37 °C in a
5 % carbon dioxide atmosphere and 100 % nominal
humidity. The doubling time was 12 h and the modal
chromosome number was 21 2. CHO cell line is par-
ticularly useful for this kind of studies because of its stable
karyotype, short cell cycle and its high plating efficiency.
ð1 ꢁ M1Þ þ ð2 ꢁ M2Þ þ ð3 ꢁ M3Þ
PRI =
100
where M1, M2, and M3 are the proportions of first, second
and third generations of mitotic cells respectively. To
evaluate the MI, cytogenetic preparations stained by
Giemsa were analyzed in a light microscope at 4009
magnification. MI was expressed as number of metaphases
per 1,000 nuclei analyzed. Scoring for chromosomal
damage was undertaken blind with coded slides. A mini-
mum of 100 metaphases per culture were scored for
chromosomal aberrations. Chromosomal aberrations were
classified as chromatid-type gaps, chromatid-type breaks,
chromatid-type exchanges, chromosome-type gaps, chro-
mosome-type breaks, chromosome-type exchanges and
isolocus events (which include isochromatid and isolocus
breaks when these cannot be distinguished), as described
by Savage (Savage, 1976). For the chromosome aberration
assay the number of aberration-bearing cells (excluding
gaps) was utilized for statistical analyses.
Chromosomal aberration assays
Test compound treatments of Chinese hamster CHO cells
were performed in the absence of rat liver metabolism. The
study was designed to comply with the experimental
methods indicated in the OECD Guidelines for the testing
of chemicals No. 487 (Draft June 2004). Following dose-
range finding experiments, the assay was performed using
dose levels of 200, 100 and 50 lM and a 3-h treatment
time. Solvent-treated cells served as negative control. At
the end of treatment (3 h), cultures were washed twice with
a PBS solution and re-incubated at 37 °C in fresh complete
culture medium for further 18 h (*1.5 cell cycle). Cultures
set up for analysis of PRI received also BrdU at 9.8 mM to
differentiate sister chromatids. To study antioxidant activ-
ity of compounds 11 and 15, cells cultures were pretreated
15 min before H2O2 (200 lM) treatment. At the end of
treatment (2 h), cultures were washed twice with a PBS
solution and re-incubated at 37 °C in fresh complete cul-
ture medium for further 18 h (*1.5 cell cycle). Colcemid
at 0.27 mM was added during the last 3 h of culture to
accumulate cells in metaphase. Hypotonic shock was
induced by 1 % trisodium citrate solution for 10 min. Cell
suspension was fixed in a mixture of methanol and glacial
acetic acid (v/v 3:1) followed by three washes. Cytogenetic
preparations for analyses of chromosomal aberrations and
mitotic indices were stained with an aqueous solution of
Giemsa 1 %. The fluorescence-plus-Giemsa (FPG) tech-
nique (Perry and Wolff, 1974) was used for sister chro-
matid differentiation (SCD) staining. Slides were stained
Conclusions
In this work a new and efficient procedure for the forma-
tion of the pyrogallol moiety was described. In particular,
3,4,5-trihydroxyphenethyl alcohol (11) and its methylether
derivative (15) were synthesized starting from an easily
available starting material. The environmental impact of
the whole process was reduced by the use of DMC as green
reactant and solvent, where possible. Furthermore, 3,4,5-
trihydroxyphenethyl alcohol and its methylated derivative
were tested for their antioxidant activity and toxicity,
showing interesting properties that could be applied in
pharmaceutical and medicine. From the biological point of
view, the presence of the methyl ether group on the side
chain (compound 15) does not modify cell viability com-
pared with compound 11. According to biological tests
performed on compound 11 and 15 the presence of a
methyl ether moiety improve the protection against H2O2
damage.
Acknowledgments The work described in this article has been
partially supported by ‘‘Ministero dell’Universita‘ e della Ricerca,
Direzione Generale per le strategie e lo sviluppo dell’internazional-
izzazione della ricerca scientifica e tecnologica’’ and ‘‘CRA-ING
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