Food and Chemical Toxicology 90 (2016) 10e17

Contents lists available at ScienceDirect

Food and Chemical Toxicology

journal homepage: www.elsevier.com/locate/foodchemtox

Modulating effect of simvastatin on the DNA damage induced by

doxorubicin in somatic cells of Drosophila melanogaster
P.C. Orsolin a, R.G. Silva-Oliveira a, J.C. Nepomuceno a, b, *
a
Universidade Federal de Uberla^ndia, Instituto de Genetica e Bioquímica, Bloco 2E, Campus Umuarama, Uberla ^ndia, Minas Gerais, Brazil
b rio de Patos de Minas, Laborato
Centro Universita rio de Citogenetica e Mutag^
enese, Patos de Minas, Minas Gerais, Brazil

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

Article history: Simvastatin is an antilipemic drug that promotes inhibition of HMG-CoA reductase. Simvastatin can also
Received 3 November 2015 inhibit the formation of other products, such as isoprenoids, conferring additional benefits to this drug,
Received in revised form which include antiproliferative, anti-invasive and pro-apoptotic effects. This study was carried out with
15 January 2016
the aim of evaluating the mutagenic/recombinogenic effect of simvastatin as well as the possible
Accepted 26 January 2016
Available online 29 January 2016
modulatory effects of this statin on the DNA damage induced by doxorubicin (DXR). This analysis was
performed using the somatic mutation and recombination test (SMART) in Drosophila melanogaster. To
study these effects, larvae descendants of both crosses (ST and HB) were chronically treated with five
Keywords:
Simvastatin
concentrations of simvastatin, separately and in association with DXR. The results revealed no muta-
Doxorubicin genic/recombinogenic effect of simvastatin for any of the concentrations tested. A modulating effect of
Drosophila melanogaster simvastatin was also observed on DNA damage induced by DXR. The reduction of total mutant frequency
Antigenotoxicity was observed for spots from descendants of both crosses, but the inhibition was more effective in de-
SMART scendants from the standard cross (ST). It is believed that this modulating effect is mainly associated with
Wing spot assay the antioxidant activity of this class of drugs, although this parameter has not been directly assessed in
this study.
© 2016 Elsevier Ltd. All rights reserved.

1. Introduction mechanisms: a decrease in the endogenous synthesis of cholesterol

Simvastatin is an antilipemic drug of the statin class, which acts
to reduce plasma cholesterol levels and, consequently, lowers the
risk of atherosclerosis and myocardial infarction (Sparrow et al.,
2001; Tang et al., 2006; Ishikawa et al., 2014). Statins were first
derived from the fungi Penicillium citrinum and its mechanism of
action is inhibition of 3-hydroxy-3-methylglutaryl-coenzyme A
reductase (HMG CoA reductase), an enzyme present in the cell's
endoplasmic reticulum, responsible for the conversion of HMG-CoA
to mevalonate in the cholesterol biosynthetic pathway (Hindler
et al., 2006; Saito et al., 2008). The main point of limitation and
control in intracellular cholesterol metabolism occurs precisely at
this stage, and is largely influenced by the degree of activity of this
enzyme (Marie et al., 2008). Inhibition of HMG-CoA reductase
promotes reduction of LDL cholesterol through two principal
^ndia, Instituto de Gene
* Corresponding author. Universidade Federal de Uberla -
tica e Bioquímica, Bloco 2E, Campus Umuarama, Uberla^ndia, Minas Gerais, Brazil.
Tel.: þ55 34 3823 0169; fax: þ 55 34 3821 0300.
E-mail address: nepomuceno@ufu.br (J.C. Nepomuceno).
and, simultaneously, an increase in receptor synthesis for LDL
cholesterol in hepatic cells, which increases the clearance thereof
(Fonseca, 2005).
There are striking pharmacokinetic differences between the
statins currently available on the market, including variations in the
coefficient of hydrophilicity, hepatic pathway, plasma half-life and
efficacy in lowering lipid profiles. Statins also differ regarding in-
teractions with other drugs that use the same pathway (Fonseca,
2005; Schachter, 2005). In this context, although all statins have
the same mechanism of action, there are differences in the effec-
tiveness of responses observed for each statin individually.
With respect to the chemical structure of statins, there are open
or closed lactone ring forms (which makes them structural similar
to HMG-CoA), constituting the active portion of this class of drugs.
Simvastatin, specifically, has a closed lactone ring, and is considered
a pro-drug activated in vivo by chemical or enzymatic reaction
(Campo and Carvalho, 2007; Banzato, 2013). In the process of
metabolism, the lactone form is converted to the active form hy-
droxy acid (Chan et al., 2003; Marinho et al., 2012.). Simvastatin is
derived from 2'-methylated lovastatin, a compound isolated from

http://dx.doi.org/10.1016/j.fct.2016.01.022
0278-6915/© 2016 Elsevier Ltd. All rights reserved.
 P.C. Orsolin et al. / Food and Chemical Toxicology 90 (2016) 10e17
the first fungi product, compactin, and is therefore considered a
semisynthetic statin (Levinski and Brown, 2007).
Besides the main action of inhibiting the synthesis of mevalo-
nate, with a consequent reduction in cholesterol levels, simvastatin
can also act by inhibiting the formation of other products, such as
isoprenoids (Demierre et al., 2005), including farnesyl and ger-
anylgeranyl groups, which bind to various proteins (superfamily
Ras/Rho) via prenylation, during the post-translational process
(Edwards and Ericsson, 1999; Yamashita et al., 2008), which gives
the drug additional benefits (pleiotropic). Such benefits include
antiproliferative, anti-invasive, pro-apoptotic and antitumor effects
(Collisson et al., 2002; Liao, 2002; Chan et al., 2003; Sleijfer et al.,
2005; Borahay et al., 2014). According to Ishikawa et al. (2014)
the antiproliferative effect of simvastatin is one of the most sig-
nificant, being greater than the effect of other statins such as flu-
vastatin and atorvastatin. The antioxidant action of statins has also
been reported in previous studies (Shishehbor et al., 2003;
Mennickent et al., 2008; Gamaleldin et al., 2015).
The extensive knowledge about the genetics of Drosophila mel-
anogaster and the long trial experience with this organism have
made it the primary tool for research on genetic mutation and
toxicology (Sarıkaya and Memmi, 2013). The SMART (Somatic
mutation and recombination test) assay conducted using D. mela-
nogaster, is a quick somatic test system, used to detect substances
that can cause changes in DNA and is also widely used to detect
antimutagenic/antirecombinogenic substances (Graf and Singer,
1992; Graf et al., 1998). This bioassay is based on the premise
that, during the embryonic development of D. melanogaster, groups
of cells proliferate mitotically to differentiate into the adult fly body
structures. If there are genetic changes in the imaginal disc cells, a
mutant cell clone will be formed and detected as a smear of mu-
tants by the wings of the adult fly (Guzman-Rico  n and Graf, 1995).
The analysis of these genetic alterations determines the phenotypic
expression of the marker genes mwh or flr3 responsible for changes
in the trichomes (Graf et al., 1984).
Considering the above, the present work was carried out with
the main objective of assessing, through the SMART assay, the
mutagenic/recombinogenic effect of simvastatin alone, as well as
the possible modulatory effects of statins on DNA damage induced
by doxorubicin (DXR). DXR is an antitumor drug of the anthracy-
cline class, which promotes intercalation with the DNA molecule
and inhibition of topoisomerase II (Islaih et al., 2005), and the
production of free radicals (Nascimento and Martins, 2005;
Kaiserova  et al., 2006). Antineoplastic DXR is a powerful and
effective chemotherapeutic agent in the treatment of several types
of tumors, but its use is limited due to its cardiotoxicity (Ewer and
Ewer, 2015).
2. Material and methods
2.1. Chemical agents
The substance tested was simvastatin (Sinvastacor®), CAS
79902-63-9, batch CV6653, with a molecular formula of C25H38O5
and a molecular weight of 418.57 g/mol, produced by Sandoz do
^utica, Cambe
Brasil Indústria Farmace , Parana. Five concentrations
of simvastatin were prepared for use in the experiment: 12.5; 25;
50; 100 and 200 mM. The chemical structure of the drug tested is
shown in Fig. 1.
Doxorubicin (Adriblastina®, CAS 25316-40-9, batch 3PL0341,
registered, imported and distributed by the laboratory Pfizer,
Guarulhos, Sa~o Paulo) was used as a positive control. Doxorubicin,
with a molecular formula of C27H29NO11-HCl and a molecular
weight of 580 g/mol, was used at a concentration of 0.125 mg/mL.
The negative control used as a reference and for dilution of the
11
Fig. 1. Chemical structure of simvastatin.
other substances was 5% ethanol. All of the dilutions were prepared
immediately before use. The use of DXR as a positive control in our
work, as well as the concentrations used, was based on studies
done previously, which demonstrated generation of reactive oxy-
gen species and induction of homologous recombination in D.
melanogaster.
2.2. Somatic mutation and recombination test (SMART) in
Drosophila melanogaster
2.2.1. Stock lineages and crosses
Three mutant lineages of D. melanogaster were used: mwh, flr3
and ORR, which possess the genetic markers multiple wing hairs
(mwh, 3e0.3) and flare3 (flr3, 3e38.8). More detailed information
about the genetic symbols and descriptions of these linkages can be
found in Lindsley and Zimm (1992).
These linkages were submitted to two crossing schemes:
a) Standard (ST) Cross, in which virgin females flr3/In(3 LR)TM3,
ri pp sep I(3)89Aa bx34e and Bds were crossed with males mwh/
mwh (Graf et al., 1989);
b) High Bioactivation (HB) Cross, in which virgin females ORR/
ORR; flr3/In(3 LR)TM3, ri pp sep I(3)89Aa bx34e and Bds were
crossed with males mwh/mwh (Graf and Van Schaik, 1992).
The use of these two types of crosses enables an evaluation of
the mutagenic and anti-mutagenic agents, of direct and indirect
action, according to the basal and elevated levels of the metabo-
lizing enzymes of cytochrome P450. This is possible because the
lineage ORR (employed in the HB cross) was created with the aim of
increasing the performance of the referred to test in the case of
activation of promutagens dependent on activation via cytochrome
P450 (Andrade and Lehmann, 2003).
From these crosses two types of descendents were obtained:
marked trans-heterozygous (MH, mwh þ/þ flr3), which have wings
with smooth edges, and balancer heterozygous (BH, mwh þ/þ TM3,
Bds), which have serrated wings (Guzm an-Rinco n and Graf, 1995).
2.2.2. Treatments
The collection of eggs of the descendents of the two crosses
described previously, ST and HB, was conducted over a period of
8 h, in vials containing solid agar base and a layer of biological yeast
supplemented with sucrose. After 72 ± 4 h, third stage larvae were
washed with reverse osmosis water, collected, and placed in glass
vials containing 1.5 g of instantaneous mashed potato (HIKARI®). To
each tube, 5 mL of simvastatin (in the concentrations 12.5; 25; 50;
100 and 200 mM) was added, diluted in 5% ethanol, alone and in
association with DXR. The association between DXR and simva-
statin occurred in a co-treatment system.
These larvae were submitted to a chronic treatment, over a
12 P.C. Orsolin et al. / Food and Chemical Toxicology 90 (2016) 10e17
period of approximately 48 h, during which time they go through
the pupal stage. The larvae were counted (in groups of 100) before
distribution into a series of vials, enabling the toxicity assays to be
performed. The number of surviving flies per treatment provided
an indicator of the toxicity of the compounds. All of the compounds
were tested in duplicate (independent series), conducted at
25  C ± 1  C and relative humidity of approximately 60%.
2.2.3. Preparation of the slides and microscopic analysis
After they hatched, the adults emerging from both crosses,
possessing trans-heterozygous (mwh þ/þ flr3) and balancer het-
erozygous (mwh þ/þ TM3, Bds) genotypes, were collected and
preserved in 70% ethanol. The wings were then removed (with the
aid of entomological forceps) and mounted on coded slides con-
taining Faure solution (50 mg of gum arabic, 30 g of chloral hydrate,
30 mL of glycerol, 50 mL of water). These slides were analyzed
under a light microscope, at a magnification of 400, with the aim
of identifying mutant spots on different sections of the wings.
2.2.4. Statistical analysis
The Kastenbaum and Bowman (1970) conditional binominal
test, at a significance level of 5%, was used to compare the fre-
quencies of each type of spot (small single, large single or twin) and
the total frequency of spots per fly, for each treatment, with this
comparison done in pairs (negative control vs. simvastatin; DXR
alone vs. simvastatin þ DXR). The multiple-decision procedure
described by Frei and Würgler (1988) was used to judge the
response as positive, weakly positive, negative or inconclusive. All
of the results with a weak positive diagnosis were analyzed with
the nonparametric U-test by Mann, Whitney and Wilcoxon (Frei
and Würgler, 1995). The percentage of inhibition of simvastatin
was calculated using the frequency of clones per 105 cells, corrected
by the control, as follows: [(DXR alone e Simvastatin þ DXR)/DXR
alone  100] (Abraham, 1994).
Based on the frequency of induction of the mutant spots (per
105 cells per division), the recombinogenic activity was calculated
by comparing the pattern of frequency of clones obtained from the
MH and BH descendents, as follows: Frequency of mutation
(FM) ¼ frequency of clones in the BH descendents/frequency of
clones in the MH descendents. Frequency of recombination (FR) ¼ 1
e frequency of mutation (FM) (Rezende et al., 2011).
3. Results
In this study all compounds were tested in chronic treatments
(approximately 48 h) on larvae descendants of the ST and HB
crosses. All of the compounds were tested in two different exper-
iments, and the results were gathered for the purpose of statistical
analyses, after checking that there was no heterogeneity in the
samples.
Before the analysis using the SMART assay, simvastatin was
submitted to a toxicity test in D. melanogaster, with the aim of
evaluating the percentage of survival of the individuals treated
with the different concentrations used. The toxicity is determined
based on the number of larvae that do not reach the adult phase.
This analysis is crucial since the reduction in the percentage of
larval survival is an indication that the compounds affected the
development of the larvae and also that the number of adults
emerging should be high enough to allow for the subsequent
treatments (Demir et al., 2013).
Concentrations used in this test were initially delineated based
on a study carried out by Spindler et al. (2012), using D. mela-
nogaster. Once the concentrations are established, the larvae, de-
scendents of the standard and high bioactivation crosses, were
submitted to a chronic treatment (for approximately 48 h) with the
substances tested. After this period, the number of flies that
hatched in each treatment scheme was counted, and this number
was then used to calculate the rates of survival after the exposure.
In this manner, the survival curves were established based on
the initial analysis of toxicity, including the percentage of flies that
hatch when treated with simvastatin alone and in association with
doxorubicin (Fig. 2). The survival percentages of both the controls
were also identified: DXR and 5% ethanol. As can be observed, the
toxic effect of simvastatin was not identified for any of the con-
centrations analyzed, not even at the highest concentration tested,
since the survival percentage for all of the concentrations was
above 85% (both in the descendents of the ST cross and the HB
cross). This is why the five concentrations tested - 12.5; 25; 50; 100
and 200 mM - were used for the SMART assay.
The results obtained in the test to detect somatic mutation and
recombination in D. melanogaster, for the marked trans-
heterozygous descendents (MH) and the balancer heterozygous
descendents (BH) of the standard (ST) cross, treated alone and co-
treated with doxorubicin, are presented in Table 1. As expected, the
positive control (DXR) promoted a statistically significant increase
(p < 0.05) in all of the mutant spot categories (single, large, twin
and total number of spots), when compared to the negative control,
which confirms its strong activity, above all, in relation to recom-
bination, confirmed after analysis of the balancer heterozygous
descendants.
In the treatment with simvastatin, none of the five concentra-
tions tested (12.5; 25; 50; 100 and 200 mM) showed an increase in
total frequency of spots (p > 0.05), when compared to the negative
control, which would suggest the absence of a mutagenic/recom-
binogenic effect for this drug. However, upon evaluating the de-
scendants treated simultaneously with simvastatin and
doxorubicin, it was observed that, in all the concentrations tested,
simvastatin reduced the frequency of single spots and total number
of spots (when compared to the positive control alone). The per-
centage of inhibition ranged from 67.07% to 87.55%, and these
values were obtained, respectively, for the concentrations of 25 mM
and 100 mM. The effect, therefore, was not dose dependent.
Once this modulating effect was identified, the balancer het-
erozygous descendants (mwh/TM3) were analyzed, with the aim of
identifying the percentages of mutagenic and recombinogenic
events. The comparison between the frequencies of spots of MH
and BH individuals revealed that recombination is the main
response to the treatment of DXR alone (50.45%). Recombination,
however, was not the main event related to the mutant spots
produced by DXR in association with simvastatin (for most of the
concentrations). In the concentrations 12.5, 25, 50, 100 and
200 mM þ DXR, 40.82%, 53.05%, 34.01%, 31.82% and 32.17% of the
spots were due to recombinogenetic changes, respectively. There-
fore, these data indicate that simvastatin, in the presence of basal
levels of cytochrome P450, significantly reduces the number of
mutant spots, altering (reducing) the recombinogenetic profile of
doxorubicin.
The results for the descendents (MH and BH) of the high bio-
activation (HB) cross, treated alone and in association with doxo-
rubicin are presented in Table 2. Once again, a significant increase
(p < 0.05) was found in the total frequency of spots identified in the
positive control, when compared to the frequency of the negative
control. DXR, therefore, induced the appearance of mutant spots,
presenting primarily recombinogenic activity (69.92%).
In the treatment with simvastatin, the results revealed no dif-
ference between the total frequency of mutant spots and the fre-
quency identified in the negative control (p > 0.05) for the five
concentrations tested. These results suggest, therefore, that, even
after intense metabolic biotransformation, simvastatin does not
possess mutagenic and/or recombinogenic effect in D.
 P.C. Orsolin et al. / Food and Chemical Toxicology 90 (2016) 10e17 13

Simvastatin
100

90

Survival (%)
80
ST
HB
70

60

50
5% Ethanol 12,5 μM 25 μM 50 μM 100 μM 200 μM

Concentrations

Simvastatin + DXR
100

90
Survival (%)

80
ST
70
HB
60

50
DXR (0,125 12,5 μM 25 μM 50 μM 100 μM 200 μM
mg/mL) +DXR +DXR +DXR +DXR +DXR

Concentrations
Fig. 2. Percentage of survival of flies after metamorphosis of larvae 72 h treated with different concentrations of simvastatin, alone and in combination with DXR.

melanogaster. demonstrated here corroborate previous results, also identified

While the analysis of descendants co-treated with simvastatin
and doxorubicin show that in the highest concentration tested,
200 mM, simvastatin significantly reduced (p < 0.05) the total fre-
quency of spots in comparison with the frequency observed in the
individuals treated only with DXR (positive control). In the other
concentrations no statistically significant reductions were observed
in the total frequency of spots.
The results also reveal that simvastatin, at a concentration of
200 mM, in the descendants of the HB cross, reduced the frequency
of spots by 65.48%. With regard to the proportion of spots obtained
by mutation and recombination, it was observed that 64.52% of the
mutant spots are related to recombinogenetic alterations. There-
fore, these data suggest that simvastatin, although it significantly
reduced the number of mutant spots at the highest concentration
tested, did little to alter the recombinogenetic profile of DXR, after
intense metabolic biotransformation.
4. Discussion
The present study investigated the mutagenic and recombino-
genic action of simvastatin, alone, as well as the possible modu-
lating effects of this statin on damage to DNA induced by
doxorubicin in the somatic cells of D. melanogaster. The effects
using the SMART assay in D. melanogaster, performed with two
synthetic statins (atorvastatin and rosuvastatin), in our laboratory,
and reported in a previous publication (Orsolin et al., 2015).
In the research cited above, the results demonstrate the absence
of a mutagenic effect for atorvastatin and rosuvastatin and a
modulating effect on the damage to DNA induced by DXR. This
effect was found in all the concentrations tested in the descendents
of the ST and HB crosses treated with rosuvastatin, and only in
descendents of the HB cross treated with atorvastatin.
In a study carried out by Peron et al. (2008), with the aim of
evaluating the cytotoxicity and mutagenicity of simvastatin in the
bone marrow cells of Wistar rats, treated in vivo, and in the meri-
stematic cells of the root of Allium cepa L., it was also found that
simvastatin does not present cytotoxic action, nor mutagenic effect,
since it did not increase the number of chromosome aberrations.
Under the present experimental conditions it was observed that
simvastatin, in addition to not presenting a mutagenic/recombi-
nogenic effect, presented a modulating response to the activity of
doxorubicin. This response was observed in the descendants of
both standard and high bioactivation crosses. Although inhibition
was more effective in the descendents of the ST cross. The differ-
ences between the two crosses, ST and HB, are related to the levels
of cytochrome P450.
14 P.C. Orsolin et al. / Food and Chemical Toxicology 90 (2016) 10e17

Table 1
Summary of results obtained in the marked trans-heterozygous descendants (MH) and balancer-heterozygous (BH) of Drosophila melanogaster derived from the standard (ST)
cross treated with different simvastatin concentrations (separately and in combination with DXR), positive control (DXR 0.125 mg/mL) and negative control (5% ethanol).

Treatments Number Spots per fly (N0 of spots); statistical diagnosisa Spots with Frequency of clone formation/ Recombination
of flies mwh clonec 105 cells per cell divisiond (%)
(N) (n)
DXR Simvastatin Small single Large single Twin Total Observed Control Inhibitione
(mg/mL) (mM) ls)b
(1e2 ce (>2 cels)b m¼5 spots corrected (%)
m¼2 m¼5 m¼2

mwh/flr3

0 0 60 0.38 (23) 0.02 (1) 0.00 (0) 0.40 (24) 24 0.82

0 12.5 60 0.33 (20)- 0.00 (0)i 0.03 (2)i 0.37 (22)- 22 0.75 0.07
0 25 60 0.32 (19)- 0.03 (2)i 0.00 (0)i 0.35 (21)- 21 0.72 0.10
0 50 60 0.38 (23)- 0.07 (4)i 0.00 (0)i 0.45 (27)- 27 0.92 0.10
0 100 60 0.40 (24)- 0.02 (1)i 0.00 (0)i 0.42 (25)- 25 0.85 0.03
0 200 60 0.47 (28)i 0.00 (0)i 0.02 (1)i 0.48 (29)- 27 0.92 0.10
0.125 0 60 1.65 (99)þ 0.20 (12)þ 0.10 (6)þ 1.95 (117)þ 97 3.31 2.49 50.45
0.125 12.5 60 0.43 (26)* 0.22 (13)i 0.08 (5)i 0.73 (44)* 43 1.47 0.65 40.82 73.90
0.125 25 60 0.53 (32)* 0.13 (8)i 0.13 (8)i 0.80 (48)* 48 1.64 0.82 53.05 67.07
0.125 50 60 0.42 (25)* 0.22 (13)i 0.10 (6)i 0.73 (44)* 43 1.47 0.65 34.01 73.90
0.125 100 60 0.33 (20)* 0.17 (10)i 0.07 (4)i 0.57 (34)* 33 1.13 0.31 31.86 87.55
0.125 200 60 0.37 (22)* 0.18 (11)i 0.15 (9)i 0.70 (42)* 42 1.43 0.61 32.17 75.50
mwh/TM3
f
0 0 40 0.28 (11) 0.00 (0) 0.28 (11) 11 0.56
0.125 0 40 0.68 (27)þ 0.13 (5)þ 0.80 (32)þ 32 1.64 1.08
0.125 12.5 40 0.43 (17)i 0.00 (0)* 0.43 (17)* 17 0.87 0.31
0.125 25 40 0.38 (15)* 0.00 (0)* 0.38 (15)* 15 0.77 0.20
0.125 50 40 0.45 (18)i 0.03 (1)i 0.48 (19)* 19 0.97 0.41
0.125 100 40 0.38 (15)* 0.00 (0)* 0.38 (15)* 15 0.77 0.20
0.125 200 40 0.48 (19)i 0.00 (0)* 0.48 (19)* 19 0.97 0.41

Marker-trans-heterozygous flies (mwh/flr3) and balancer-heterozygous flies (mwh/TM3) were evaluated.

*p  0.05 vs. DXR only.
a
Statistical diagnoses according to Frei and Würgler (1995). U-test, two sided; probability levels: -, negative; þ, positive; i, inconclusive; p  0.05 vs. negative control (5%
ethanol).
b
Including rare single flr3 spots.
c
Considering the mwh clones for the single spots and mwh for the twin spots.
d
Frequency of clone formation: clones/flies/48,800 cells (without size correction).
e
Calculated as [(DXR alone e Statin þ DXR/DXR alone)  100], according to Abraham (1994).
f
Balancer chromosome TM3 does not carry the flr3 mutation and recombination is suppressed, due to the multiple inverted regions in these chromosomes.

The HB cross used the lineage ORR, which presents chromo-
somes 1 and 2 from the lineage Oregon R, which is resistant to DDT,
and exhibits high expression of the cytochrome P450 enzymes
€lich and Wrügler, 1989; Graf and Van Schaik, 1992). Whereas
(Fro
the descendants of the ST cross have basal levels of cytochrome
P450. According to Andrade and Lehmann (2003), the lineage ORR
was developed with the aim of increasing the performance of the
SMART assay for the activation of promutagens that depend on
activation via cytochrome P450.
Simvastatin is a prodrug, administered in the form of an active
lactone, requiring hepatic transformation for conversion into b-
hydroxy acid, its active metabolite (Sabina et al., 2012; Gazzerro
et al., 2012). Most of the statins, including simvastatin, are metab-
olized by enzymes from the cytochrome P450 system (CYP3A4)
(Gazzerro et al., 2012). CYP34A is the most abundant isoenzyme of
cytochrome P450 in the liver and small intestine; it acts on the
oxidative metabolism of around 50% of all the drugs available on
the market (Granvil et al., 2003). The CYP3A4 of mammals is
analogous to the family CYP6 in Drosophila (Tijet et al., 2001).
Through these systems of biotransformation, like cytochrome
P450, biologically inactive substances can be introduced into the
body and converted into active metabolites due to alterations in
their structure. Although the purpose of deep biotransformation of
the xenobiotics is detoxification, the metabolite is not always less
toxic than the compound itself (Guecheva and Henriques, 2003).
The process of biotransformation can generate toxic intermediaries,
electrophilics, mutagenics or carcinogenics (Hodgson and Rose,
2007).
In the present study, although biotransformation did not
generate active metabolites with a mutagenic/recombinogenic ef-
fect, the product resulting from the metabolic biotransformations
of simvastatin, when associated with doxorubicin (in a co-
treatment system), reduced the total number of mutant spots.
However, with less effective inhibition (observed only at the
highest concentration) to that obtained without metabolic trans-
formation (observed at all of the concentrations tested).
The mechanisms by which simvastatin reduces the damage to
DNA induced by DXR were not directly evaluated in this study.
However, the use of the co-treatment system enables an analysis of
the effect of simvastatin on stages that occur before the harm
induced by DXR, enabling an evaluation of its modulating effect. As
such, it can be assumed, based on the results obtained in the pre-
sent study, that the active metabolites of simvastatin, resulting
from the process of biotransformation, are capable of interacting
directly with the active groups of the DXR, altering the inhibition
response, as seen in the descendants of the standard (ST) cross. This
is a possibility, though one that cannot be explained precisely in the
present study, since it is not possible to identify how this interac-
tion between the two substances occurs.
The results for the individuals treated with DXR confirmed the
recombinogenic effect of this drug. With regard to the action
mechanism, doxorubicin binds strongly to DNA due to its ability to
intercalate between base pairs. When intercalating in a DNA
molecule, DXR promotes breakage in the molecule, inhibiting the
synthesis of DNA and RNA (Craig and Stitzel, 2005; Kaiserova  et al.,
2006), while also promoting the production of free radicals
(Nascimento and Martins, 2005). According to Lehmann et al.
(2003), the intrinsic link between inhibition of topoisomerases,
 P.C. Orsolin et al. / Food and Chemical Toxicology 90 (2016) 10e17 15

Table 2
Summary of results obtained in the marked trans-heterozygous descendants (MH) and balancer-heterozygous (BH) of Drosophila melanogaster derived from the high bio-
activation (HB) cross treated with different simvastatin concentrations (separately and in combination with DXR), positive control (DXR 0.125 mg/mL) and negative control (5%
ethanol).

Treatments Number Spots per fly (N0 of spots); statistical diagnosisa Spots with Frequency of clone formation/ Recombination
of flies mwh clonec 105 cells per cell divisiond (%)
(N) (n)
DXR Simvastatin Small single Large single Twin Total Observed Control Inhibitione
(mg/mL) (mM) ls)b
(1e2 ce ls)b
(>2 ce m¼5 spots m ¼ 2 corrected (%)
m¼2 m¼5

mwh/flr3

0 0 60 1.10 (66) 0.03 (02) 0.02 (1) 1.15 (69) 69 2.36

0 12.5 60 1.12 (67)- 0.08 (5)i 0.02 (1)i 1.22 (73)- 71 2.42 0.07
0 25 60 1.05 (63)- 0.08 (5)i 0.03 (2)i 1.17 (70)- 70 2.39 0.03
0 50 60 1.30 (78)- 0.08 (5)i 0.00 (0)i 1.38 (83)- 82 2.80 0.44
0 100 60 1.20 (72)- 0.07 (4)i 0.05 (3)i 1.32 (79)- 79 2.70 0.34
0 200 60 1.32 (79)- 0.03 (2)i 0.02 (1)i 1.37 (82)- 82 2.80 0.44
0.125 0 60 1.48 (89)- 2.20 (132)þ 0.10 (6)i 3.78 (227)þ 225 7.68 5.33 69.92
0.125 12.5 60 1.43 (86) ns 1.38 (83)* 0.13 (8)i 2.95 (177) ns 173 5.91 3.55
0.125 25 60 1.48 (89) ns 1.35 (81)* 0.07 (4)i 2.90 (174) ns 174 5.94 3.59
0.125 50 60 1.27 (76) ns 1.55 (93)* 0.25 (15)* 3.07 (184) ns 182 6.22 3.86
0.125 100 60 1.40 (84) ns 1.05 (63)* 0.25 (15)* 2.70 (162) ns 161 5.50 3.14
0.125 200 60 1.22 (73) ns 0.77 (46)* 0.08 (5)i 2.07 (124)* 123 4.20 1.84 64.52 65.48
mwh/TM3
f
0 0 40 0.38 (15) 0.00 (0) 0.38 (15) 15 0.77
0.125 0 40 0.98 (39)i 0.15 (6)i 1.13 (45)þ 45 2.31 1.54
0.125 200 40 0.73 (29)i 0.00 (0)* 0.73 (29)* 29 1.49 0.72

Marker-trans-heterozygous flies (mwh/flr3) and balancer-heterozygous flies (mwh/TM3) were evaluated.

*p  0.05 vs. DXR only, ns: not significant.
a
Statistical diagnoses according to Frei and Würgler (1995). U-test, two sided; probability levels: -, negative; þ, positive; i, inconclusive; p  0.05 vs. negative control (5%
ethanol).
b
Including rare single flr3 spots.
c
Considering the mwh clones for the single spots and mwh for the twin spots.
d
Frequency of clone formation: clones/flies/48,800 cells (without size correction).
e
Calculated as [(DXR alone e Statin þ DXR/DXR alone)  100], according to Abraham (1994).
f
Balancer chromosome TM3 does not carry the flr3 mutation and recombination is suppressed, due to the multiple inverted regions in these chromosomes.

blocking of the synthesis and replication of DNA and inducing
double breaks, associated with the fact that this latter event is one
of the main substrates for the occurrence of recombination, also
make DXR a potent recombinogenic agent. This recombinogenic
effect has already been reported in a series of previous studies
performed on the somatic cells of D. melanogaster (Lehmann et al.,
2003; Costa and Nepomuceno, 2006; Rezende et al., 2009; Orsolin
et al., 2012, 2015).
Another possible mechanism to explain the reduction in the
number of mutant spots produced by simvastatin, when associated
with DXR, could be associated with the antioxidant effect of statins.
Elbaky et al. (2006) investigated the possible protective effects of
simvastatin on the nephrotoxicity induced by adriamycin in rats
and found that the administration of simvastatin, before and con-
current with adriamycin, promoted a reduction in lipid peroxida-
tion and an increase in the activity of antioxidant enzymes.
Similarly, Elbaky et al. (2010) investigated possible protective
effects of simvastatin on the cardiotoxicity induced by doxorubicin.
These authors found that the administration of simvastatin also
significantly reduced the myocardial alterations induced by treat-
ment with DXR. These results suggest that simvastatin offers pro-
tection against cardiac damage induced by DXR, in terms of
oxidative stress.
Ungureanu et al. (2003) observed that individuals with hyper-
cholesterolemia, who received daily doses of simvastatin (10 mg/
day, for 8 months), in addition to a significant reduction in total
cholesterol, presented a significant improvement in some anti-
oxidant enzyme parameters: superoxide dismutase, glutathione
peroxidase and catalase.
In addition to the recognized antioxidant effect of simvastatin,
which may have resulted in the reduction of mutant spots induced
by DXR, this reduction may also be associated with the apoptotic
effect of this drug. In a study conducted by Yanae et al. (2011), it was
demonstrated that statins inhibit cellular proliferation and induce
apoptosis in the glioma cells of rats. An increase in the activity of
caspase-3, an enzyme with a central role in this process, was also
observed. In addition, the statins reduced the levels of Ras regu-
lated by the ERK1/2 and Akt pathways. According to the authors
cited, these findings indicate that statins can be used as anti-
carcinogenic agents in glioblastoma.
Takeda et al. (2007) also demonstrated that simvastatin is
capable of suppressing cellular survival and the invasive activity of
squamous carcinoma cells of the head and neck by activating
integrin b1 (involved in the ERK signaling cascade). These authors
also confirmed the activation of the inhibitors of CDK, p21 and p27,
and the inhibitors of caspase-3. According to Bardou et al. (2010),
statins, in the case of colorectal cancer, may counter the growth of
cancer by inducing apoptosis, by reducing the expression of anti-
apoptotic proteins like BC12 or clAP1 and by increasing the
expression of pro-apoptotic proteins, like BMP, for example.
As a result of the effects described above, statins, including
simvastatin, have been highlighted for their possible antitumor
effects. In this sense, Bardou et al. (2010) affirmed that there are
various mechanisms that could justify the antitumor effects of
statins, including the induction of apoptosis (through different
pathways), inhibition of cellular growth, in addition to angiogen-
esis and an improvement of the immune response.
In conclusion, the results reported showed that, under the
present experimental conditions, simvastatin presented a modu-
lating effect on the damage to DNA induced by doxorubicin in the
somatic cells of D. melanogaster. It is believed that this modulating
effect is associated with the apoptotic and, primarily, antioxidant
16 P.C. Orsolin et al. / Food and Chemical Toxicology 90 (2016) 10e17
activities of this class of drugs, although these effects have not been
directly evaluated in this study. It is suggested, therefore, that new
studies involving other test organisms and experimental models be
conducted with the aim of promoting a better understanding of the
modulating activity of simvastatin in relation to substances that
induce damage to DNA, such as doxorubicin.
Transparency document
Transparency document related to this article can be found
online at http://dx.doi.org/10.1016/j.fct.2016.01.022.
References
Abraham, S.K., 1994. Antigenotoxicity of coffee in the Drosophila assay for somatic
mutation and recombination. Mutagenesis 9, 383e386.
Andrade, H.H.R., Lehmann, M., 2003. Teste para detecça ~o de mutaça ~o e
recombinaç~ ao som atica em Drosophila melanogaster. In: Ribeiro, L.R.,
Salvadori, D.M.F., Marques, E.K. (Eds.), Mutage ^nese Ambiental. Ulbra, Canoas,
pp. 281e307.
Banzato, T.P., 2013. Avaliaça ~o da toxicidade reprodutiva da sinvastatina em ratos
adultos. Dissertaça ~o (Biologia Geral e Aplicada). Instituto de Biocie ^ncias, Uni-
versidade Estadual Paulista, Botucatu.
Bardou, M., Barkun, A., Martel, M., 2010. Effect of statin therapy on colorectal cancer.
Gut 59, 1572e1585.
Borahay, M.A., Kilic, G.S., Yallampalli, C., Snyder, R.R., Hankins, G.D.V., Al-Hendy, A.,
Boehning, D., 2014. Simvastatin potently induces calcium-dependent apoptosis
of human leiomyoma cells. J. Biol. Chem. 51, 35075e35086.
Campo, V.L., Carvalho, I., 2007. Estatinas hipolipe ^micas e novas tende ^ncias ter-
ape^uticas. Quím. Nova 30, 01e14.
Chan, K.K., Oza, A.M., Siu, L.L., 2003. The statins as anticancer agents. Clin. Cancer
Res. 9, 10e19.
Collisson, E.A., Carranza, D.C., Chen, I.Y., Kolodney, M.S., 2002. Isoprenylation is
necessary for the full invasive potential of RhoA overexpression in human
melanoma cells. J. Investigative Dermatol. 119, 1172e1176.
Costa, W.F., Nepomuceno, J.C., 2006. Protective effects of a mixture of antioxidant
vitamins and mineral on the genotoxicity of doxorubicin in somatic cells of
Drosophila melanogaster. Environ. Mol. Mutagen 47, 18e24.
Craig, C.R., Stitzel, R.E., 2005. Farmacologia Moderna: Com Aplicaço ~es Clínicas, 6. ed.
Guanabara Koogan, Rio de Janeiro. 815 pp.
Demierre, M.F., Higgins, P.D., Gruber, S.B., Hawk, E., Lippman, S.M., 2005. Statins and
cancer prevention. Nat. Rev. Cancer 5, 930e942.
Demir, E., Turna, F., Kaya, B., Creus, A., Marcos, R., 2013. Mutagenic/recombinogenic
effects of four lipid peroxidation products in Drosophila. Food Chem. Toxicol.
53, 221e227.
Edwards, P.A., Ericsson, J., 1999. Sterols and isoprenoids: signaling molecules
derived from the cholesterol biosynthetic pathway. Annu. Rev. Biochem. 68,
157e185.
Elbaky, N.A.A., Hammad, L.N., Atia, T.A., 2006. Protective effect of simvastatin
against adriamycin-Induced nephrotoxicity in rats; biochemical and histologi-
cal study. Egypt. J. Hosp. Med. 25, 725e739.
Elbaky, N.A.A., Ali, A.A., Ahmed, R.A., 2010. Cardioprotective effect of simvastatin on
doxorubicin -induced cardiotoxicity in rats. J. Basic Appl. Sci. 6, 1e11.
Ewer, M.S., Ewer, S.M., 2015. Cardiotoxicity of anticancer treatments. Nat. Rev.
Cardiol. 12, 547e558.
Fonseca, F.A.H., 2005. Farmacocine tica das estatinas. Arq. Bras. Cardiol. 85, 09e14.
Frei, H., Würgler, F.E., 1988. Statistical methods to decide whether mutagenicity test
data from Drosophila assay indicate a positive, negative or inconclusive result.
Mutat. Res. 203, 297e308.
Frei, H., Würgler, F.E., 1995. Optimal experimental design and sample size for the
statistical evaluation of data from somatic mutation and recombination test
(SMART) in Drosophila. Mutat. Res. 334, 247e258.
€lich, A., Würgler, F.E., 1989. New tester strains with improved bioactivation ca-
Fro
pacity for the Drosophila wing-spot test. Mutat. Res. 216, 179e187.
Gamaleldin, I.H., Fars, K.A., Sabry, M.A., Gamal, A.O., 2015. Influence of simvastatin
chronotherapy on erythrocytes nitric oxide synthase activity. Int. J. Pharmacol.
5, 448e455.
Gazzerro, P., Proto, M.C., Gangemi, G., Malfitano, A.M., Ciaglia, E., Pisanti, S.,
Santoro, A., Laezza, C., Bifulco, M., 2012. Pharmacological actions of statins: a
critical appraisal in the management of cancer. Pharmacol. Rev. 64, 102e146.
Graf, U., Abraham, S.K., Guzma n-Rinco  n, J., Würgler, F.E., 1998. Antigenotoxicity
studies in Drosophila melanogaster. Mutat. Res. 402, 203e209.
Graf, U., Frei, H., K€agi, A., Katz, A.J., Würgler, F.E., 1989. Thirty compounds tested in
the Drosophila wing spot test. Mutat. Res. 222, 359e373.
Graf, U., Singer, D., 1992. Genotoxicity testing of promutagens in the wing somatic
mutation and recombination test in Drosophila melanogaster. Rev. Int. Contam.
Ambient. 8, 15e27.
Graf, U., Van Schaik, N., 1992. Improved high bioactivation cross for the wing so-
matic and recombination test in Drosophila melanogaster. Mutat. Res. 271,
59e67.
Graf, U., Würgler, F.E., Katz, A.J., Frei, H., Juon, H., Hall, C.B., Kale, P.G., 1984. Somatic
mutation and recombination test in Drosophila melanogaster. Environ. Mutagen
6, 153e188.
Granvil, C.P., Yu, A.M., Elizondo, G., Akiyama, T.E., Cheung, C., Feigenbaum, L.,
Kraus, K.W., Gonzales, F.J., 2003. Expression of the human CYP3A4 gene in the
small intestine of transgenic mice: in vitro metabolism and pharmacokinetics of
midazolam. Cancer Lett. 167, 99e104.
Guecheva, T.K., Henriques, J.A.P., 2003. Metabolismo de xenobio  ticos: citocromo P-
450. In: Silva, J., Erdtmann, B., Henriques, J.A.P. (Eds.), Gene tica Toxicolo gica.
Alcance, Porto Alegre, pp. 223e247. Cap.11.
Guzma n-Ricon, J., Graf, U., 1995. Biomonitors and Biomarkers as Indicators of
Environmental Change. Phenunm Press, New York, pp. 169e181.
Hindler, K., Eltzschig, H.K., Fox, A.A., Body, S.C., Shernan, S.K., Collard, C.D., 2006.
Influence of statins on perioperative outcomes. J. Cardiothorac. Vasc. Anesth. 20,
251e258.
Hodgson, E., Rose, R.L., 2007. The importance of cytochrome P450 2B6 in the human
metabolism of environmental chemicals. Pharmacol. Ther. 113, 420e428.
Ishikawa, S., Hayashi, H., Kinoshita, K., Abe, M., Kuroki, H., Tokunaga, R.,
Tomiyasu, S., Tanaka, H., Sugita, H., Arita, T., Yagi, Y., Watanabe, M., Hirota, M.,
Baba, H., 2014. Statins inhibit tumor progression via an enhancer of zeste ho-
molog 2-mediated epigenetic alteration in colorectal cancer. Int. J. Cancer 135,
2528e2536.
Islaih, M., Halstead, B.W., Kadura, I.A., Li, B., Reid-Hubbard, J.L., Flick, L., Altizer, J.L.,
Thom Deahl, J., Monteith, D.K., Newton, R.K., Watson, D.E., 2005. Relationships
between genomic, cell cycle, and mutagenic responses of TK6 cells exposed to
DNA damaging chemicals. Mutat. Res. 578, 100e116.
Kaiserova , H., Den Hartog, G.J.M., Simunek, T., Schroterov a, L., Kvasnickov a, E.,
Bast, A., 2006. Iron is not involved in oxidative stress-mediated cytotoxicity of
doxorubicin and bleomycin. Br. J. Pharmacol. 149, 920e930.
Kastenbaum, M.A., Bowman, K.O., 1970. Tables for determining the statistical sig-
nificance of mutation frequencies. Mutat. Res. 9, 527e549.
Lehmann, M., Franco, A., Vilar, K.S.P., Reguly, M.L., Andrade, H.H.R., 2003. Doxoru-
bicin and two of its analogues are preferential inducers of homologous
recombination compared with mutational events in somatic cells of Drosophila
melanogaster. Mutat. Res. 539, 167e175.
Levinski, R.J., Brown, H., 2007. Statins. Too many people are taking them (and
they're doing far less good than you think). Lancet 69, 268e269.
Liao, J.K., 2002. Beyond lipid lowering: the role of statins in vascular protection. Int.
J. Cardiol. 86, 05e18.
Lindsley, D.L., Zimm, G.G., 1992. The Genome of Drosophila melanogaster, first ed.
Academic Press, San Diego. 1133pp.
Marie, I., Delafenetre, H., Massy, N., Thuillez, C., Noblet, C., 2008. Tendinous disor-
ders attributed to statins: a study on ninety-six spontaneous reports in the
period 1990-2005 and review of the literature. Arthritis Rheum. 59, 367e372.
Marinho, F.D.M., Diniz, M.M.L., Zanon, J. C. da C., Reis, I.A., Lima, A.A., Soares, C.D.V.,
2012. Influe ^ncia da temperatura em estudos de dissoluça ~o de formas farm-
ace^uticas contendo sinvastatina. Rev. Bras. Farm. 93, 38e42.
Mennickent, S.C., Bravo, M.D., Calvo, C.M., Avello, M.L., 2008. Efectos pleiotro picos
de las estatinas. Rev. Med. Chile 136, 775e782.
Nascimento, M.C.M.O., Martins, A.S., 2005. Cardiomiopatia induzida pela adriami-
cina: uma revis~ ao. Arq. Cie^ncias Saúde 12, 111e115.
Orsolin, P.C., Silva-Oliveira, R.G., Nepomuceno, J.C., 2012. Assessment of the muta-
genic, recombinagenic and carcinogenic potential of orlistat in somatic cells of
Drosophila melanogaster. Food Chem. Toxicol. 50, 2598e2604.
Orsolin, P.C., Silva-Oliveira, R.G., Nepomuceno, J.C., 2015. Modulating effect of syn-
thetic statins against damage induced by doxorubicin in somatic cells of
Drosophila melanogaster. Food Chem. Toxicol. 81, 111e119.
Peron, A.P., Felipes, J., Oliveira, A.C.S., Vicentin, V.E.P., 2008. Investigaç~ ao da cit-
otoxicidade e mutagenicidade do hipocolesteremiante sinvastatina em ce lulas
de ratos wistar e de Allium cepa L. Rev. Bras. Toxicol. 21, 93e96.
Rezende, A. A. de, Graf, U., Guterres, Z.R., Kerr, W.E., Spano  , M.A., 2009. Protective
effects of proanthocyanidins of grape (Vitisvinifera L.) seeds on DNA damage
induced by Doxorubicin in somatic cells of Drosophila melanogaster. Food Chem.
Toxicol. 47, 1466e1472.
Rezende, A. A. de, Silva, M.L.A., Tavares, D.C., Cunha, W.R., Rezende, K.C.S.,
Bastos, J.K., Lehmann, M., Andrade, H. H. R. de, Guterres, Z.R., Silva, L.P.,
Spano , M.A., 2011. The effect of the dibenzylbutyrolactoliclignan (-)-cubebin on
doxorubicin mutagenicity and recombinogenicity in wing somatic cells of
Drosophila melanogaster. Food Chem. Toxicol. 49, 1235e1241.
Sabina, A.G., Da vila, J.G., Serrano, P.S., Juanetey, C.G., Pacheco, R.M., 2012. Consid-
eraciones específicas en la prescripcio n e intercambio terape utico de estatinas.
Farm. Hosp. 36, 97e108.
Saito, A., Saito, N., Mol, W., Furukawa, H., Tsutsumida, A., Oyama, A., Sekido, M.,
Sasaki, S., Yamamoto, Y., 2008. Simvastatin inhibits growth via apoptosis and
the induction of cell cycle arrest in human melanoma cells. Melanoma Res. 18,
85e94.
Sarıkaya, R., Memmi, B.K., 2013. Detection of transfluthrin and metofluthrin geno-
toxicity in the ST cross of the Drosophila Wing Spot Test. Chemosphere 93,
238e242.
Schachter, M., 2005. Chemical, pharmacokinetic and pharmacodynamic properties
of statins: an update. Fundam. Clin. Phermacol. 19, 117e125.
Shishehbor, M.H., Brennan, M.L., Aviles, R.J., Fu, X., Penn, M.S., Sprecher, D.L.,
Hazen, S.L., 2003. Statins promote potent systemic antioxidant effects through
specific inflammatory pathways. Circulation 108, 426e431.
Sleijfer, S., Van Der Grasst, A., Planting, A.S., Stoter, G., Verweij, J., 2005. The potential
 P.C. Orsolin et al. / Food and Chemical Toxicology 90 (2016) 10e17
of statins as part of anti-cancer treatment. Eur. J. Cancer 41, 516e522.
Sparrow, C.P., Burton, C.A., Hernandez, M., Mundt, S., Hassing, H., Patel, S., Rosa, R.,
Hermanowski-Vosatka, A., Wang, P.R., Zhang, D., Peterson, L., Detmers, P.A.,
Chao, Y.S., Wright, S.D., 2001. Simvastatin has anti-inflammatory and anti-
atherosclerotic activities independent of plasma cholesterol lowering. Arterio-
sclerosis Thrombosis Vasc. Biol. 21, 115e121.
Spindler, R.S., Li, R., Dhahbi, J.M., Yamakawa, A., Mote, P., Bodmer, R., Ocorr, K.,
Willians, R.T., Wang, Y., Ablao, K.P., 2012. Statin treatment increases lifespan and
improves cardiac health in Drosophila by decreasing specific protein pre-
nylation. PLoS One 7 (6), e39581.
Takeda, I., Maruya, C.I., Shirasaki, T., Misukami, H., Takahata, T., Myers, J.N.,
Kakehata, S., Yagihashi, S., Shimkawa, H., 2007. Simvastatin inactivates b1-
integrin and extracellular signal-related kinase signaling and inhibits cell pro-
liferation in head and neck squamous cell carcinoma cells. Cancer Sci. 98,
890e899.
Tang, D., Park, H., Georgescu, S.P., Sebti, S.M., Hamilton, A.D., Galper, J.B., 2006.
Simvastatin potentiates tumor necrosis factor a-mediated apoptosis of human
17
vascular endothelial cells via the inhibition of the geranylgeranylation of RhoA.
Life Sci. 79, 1484e1492.
Tijet, N., Helvig, C., Feyereisen, R., 2001. The cytochrome P450 gene superfamily in
Drosophila melanogaster: annotation, introneexon organization and phylogeny.
Gene 262, 189e198.
Ungureanu, D., Filip, C., Artenie, A., Artenie, R., 2003. Evaluation of simvastatin
antioxidant effects. Rev. Med. Chir. Soc. Med. Nat. Iasi 107, 66e71.
Yamashita, M., Otsuka, F., Mukai, T., Otani, H., Inagaki, K., Miyoshi, T., Goto, J.,
Yamamura, M., Makino, H., 2008. Simvastatin antagonizes tumor necrosis
factor-a inhibition of bone morphogenetic proteins-2-induced osteoblast dif-
ferentiation by regulating Smad signaling and Ras/Rho-mitogen-activated
protein kinase pathway. J. Endocrinol. 196, 601e613.
Yanae, M., Tsubaki, M., Satou, T., Itoh, T., Imano, M., Yamazoe, Y., Nishida, S., 2011.
Statin induced apoptosis via the suppression of ERK1/2 and Akt activation by
inhibition of the geranylgeranyl-pyrophosphate biosynthesis in glioblastoma.
J. Exp. Clin. Cancer Res. 30, 74e82.