GENOTOXICITY AND CARCINOGENICITY

The positive response of Ty1 retrotransposition test to carcinogensis due to increased levels of reactive oxygen species generatedby the genotoxins

Martin Dimitrov • Pencho Venkov •

Margarita Pesheva

Received: 17 February 2010 / Accepted: 1 April 2010 / Published online: 17 April 2010

� Springer-Verlag 2010

Abstract In previous laboratory and environmental

studies, the Ty1 short-term test showed positive responses

(i.e. induced mobility of the Ty1 retrotransposon) to car-

cinogenic genotoxins. Here, we provide evidence for a

causal relationship between increased level of reactive

oxygen species and induction the mobility of the Ty1 ret-

rotransposon. Results obtained in concentration and time-

dependent experiments after treatment, the tester cells with

carcinogenic genotoxins [benzo(a)pyrene, benzo(a)anthra-

cene, ethylmethanesulfonate, formamide], free bile acids

(chenodeoxycholic, lithocholic acids) and metals (arsenic,

hexavelant chromium, lead) showed a simultaneous

increase in both cellular level of the superoxide anions and

Ty1 retrotransposition rates. Treatment with the noncarci-

nogenic genotoxins [benzo(e)pyrene, benzo(b)anthracen,

anthracene], conjugated bile acids (taurodeoxycholic, gly-

codeoxycholic acids) and metals (zinc, trivalent chromium)

did not change significantly superoxide anions level and

Ty1 retrotransposition rate. The induction by carcinogens

of the Ty1 mobility seems to depend on the accumulation

of superoxide anions, since the addition of the scavenger

N-acetylcysteine resulted in loss of both increased amount

of superoxide anions and induced Ty1 retrotransposition.

Increased hydrogen peroxide levels are also involved in the

induction of Ty1 retrotransposition rates in response to

treatment with carcinogenic genotoxins, as evidenced by

disruption of YAP1 gene in the tester cells. It is concluded

that the carcinogen-induced high level of reactive oxygen

species play a primary and key role in determination the

selective response of Ty1 test to carcinogenic genotoxins.

Keywords Ty1 retrotransposition � Carcinogens �Reactive oxygen species

Introduction

Ty1 elements are the most abundant class of retrotranspo-

sons in the yeast Saccharomyces cerevisiae with 32 copies

per haploid genome (Kim et al. 1998). They replicate via a

RNA intermediate which is encapsulated into virus-like

particles together with Ty1-encoded structural proteins and

the enzymes protease, integrase and reverse transcriptase.

Inside these particles, Ty1-RNA is reverse transcribed and

the copy-DNA is integrated into new locations of the gen-

ome either by integrase-mediated or to a lesser extent by

homologues recombination with genomic Ty1 elements.

The new Ty1 copy generates substantial genome instability

leading to appearance of a wide range of DNA damages,

including point mutations, deletions, inversions, amplifica-

tions and large genomic rearrangements (Garfinkel 1992).

Thus, Ty1 is a typical retrotransposon which structure and

replication cycle resemble the known retrooncoviruses. The

frequency of spontaneous Ty1 retrotransposition is low,

about 10-7/element/generation (Paquin and Williamson

1984) and is induced by stress conditions, such as nitrogen or

adenine starvation (Morrilon et al. 2000; Todeschini et al.

2005), freezing (Stamenova et al. 2008), exposure to UV

light or X-rays (Sacerdot et al. 2005). Chemical carcinogens

also activate Ty1 retrotransposition (Pesheva et al. 2005,

2008). The carcinogen-induced Ty1 retrotransposition

M. Dimitrov � M. Pesheva (&)

Sofia University Faculty of Biology,

8 ‘‘Dragan Tsankov’’ blvd, 1421 Sofia, Bulgaria

e-mail: mpesheva2000@yahoo.com

P. Venkov

Institute of Cryobiology and Food Technology,

53A ‘‘Cherni Vrah’’ blvd, 1407 Sofia, Bulgaria

123

Arch Toxicol (2011) 85:67–74

DOI 10.1007/s00204-010-0542-8

depends on the function of RAD9 gene and transit through

G1 phase of the cell cycle (Staleva and Venkov 2001). The

protein product of RAD9 check-point gene is the yeast

functional counterpart of the human repressor protein p53,

which has the function to monitor the integrity of the genome

and to delay DNA replication in G1 phase until repair has

been completed (Bertram 2001). These data show the exis-

tence of similarities in certain steps in regulation of Ty1

retrotransposition and neoplasmic differentiation of cells

and suggest that Ty1 retrotransposition might be used as a

test system for detection of carcinogens.

The Ty1 test is a short-term test for detection of car-

cinogens based on the induction the retrotransposition of a

gene-engineered oncogene-like Ty1 element (Pesheva

et al. 2005). The test response was studied after treatment

the tester cells with laboratory genotoxins and also used in

environmental monitoring studies (Pesheva et al. 2008).

The Ty1 test responded positively (e.g. with increased

retrotransposition) only after treatment the tester cells

with carcinogenic genotoxins or with extracts of envi-

ronmental samples polluted with carcinogens. The Ty1

test gave negative results with noncarcinogenic mutagens

or environmental samples polluted only with mutagens

without carcinogenic potential. This selective response of

Ty1 test to carcinogens was also proved with substances

having different mechanisms of carcinogenicity in mam-

malian cells, and the specificity of the response to

carcinogens was further evidenced by testing pairs of

laboratory genotoxins with very similar chemical struc-

ture, the one being a strong carcinogen, the second having

only mutagenic activity without being a carcinogen

(Pesheva et al. 2008). The reasons for the selective and

specific activation of Ty1 retrotransposition by carcino-

gens are not yet understood.

The survey of the literature showed that many carcin-

ogens that activate Ty1 mobility are also powerful

inducers of oxidative stress in different cells (Scandalious

2005), including S. cerevisiae (Herrero et al. 2008).

Recently, it was shown that the carcinogen-induced Ty1

retrotransposition depends on the intactness of mitochon-

dria (Stoycheva et al. 2007), which is the major source for

production of reactive oxygen species (ROS). In another

study (Stamenova et al. 2008), it was reported that the

reason for the induction of Ty1 retrotransposition fol-

lowing freezing the yeast cells is the accumulation of

ROS in frozen cells. These data suggest the involvement

of increased ROS levels in the activation of Ty1 retro-

transposition and the results of such a study are reported

in this communication.

The obtained results evidence that carcinogens induced

both generation of ROS and activation of Ty1 retrotrans-

position, while mutagens without carcinogenic potential

did not change significantly the level of ROS in

S. cerevisiae cells. The generation of high levels of ROS

seems to have a key and independent role in carcinogen-

induced Ty1 retrotransposition since the neutralization of

the burst of ROS with scavengers is accompanied with a

strong decrease of Ty1 mobility.

Materials and methods

Strains and cultivation of cells

The S. cerevisiae 551 strain (Pesheva et al. 2005) was used

as a tester strain in the Ty1 retrotransposition assay. It has a

Ty1 element marked with the indicator gene HIS3AI con-

structed by Curcio and Garfinkel (1991), the mutation

sec53 that increases the permeability of cells and additional

auxotrophic markers. The isogenic strain S. cerevisiae

551yap1D (generously made available by T. Stoycheva)

has a disrupted YAP1 gene and was constructed by trans-

formation of 551 cells with the yap1:: hisG-URA3-hisG

cassette. Cells were cultivated at 30�C in YEPD liquid

medium to exponential phase of growth corresponding to a

density of 4–6 9 107 cells/ml. Laboratory genotoxins were

added to culture aliquots for 30 min in presence or absence

of metabolic activation by S9 microsomal hepatic fraction.

In experiments with scavengers of ROS, N-acetylcysteine

(60 mM) was added 30 min before the treatment of cells

with genotoxin.

Ty1 retrotransposition test

Each successful transposition event of the marked Ty1 in

strain 551 requires transcription, splicing the artificial AI

intron, reverse transcription and insertion of the resulting

copy-DNA into a new location of the genome which gives

rise to one histidine prototrophic colony on selective

medium. Thus, the number of His? transformants is a

quantitative measure for the frequency of retrotransposition

of the marked Ty1.

The Ty1 retrotransposition test was performed as

described (Pesheva et al. 2005). Treated and control cells

were collected, suspended in fresh YEPD medium and

cultivated at 20�C for 16 h to complete the initiated ret-

rotransposition events. Appropriate dilutions of cells were

plated to determine survivals (on YEPD) and number of

transposants (on SC-histidine). Mean rates of retrotrans-

position were determined by the equation of Drake (1970),

and the average values ±SD from 5 to 10 repetitions of

each experiment were calculated. In the tables, results are

also presented as ‘‘fold increase’’ of Ty1 retrotransposition

related to the control sample taken as fold increase of 1.0.

An increase higher than 2.0 is considered as a positive

result of the assay (Pesheva et al. 2005).

68 Arch Toxicol (2011) 85:67–74

123

Quantitative assay for superoxide anions

We used an assay for superoxide anions (O2-) determina-

tion (Sutherland and Learmont 1997) adapted to S. cerevi-

siae cells (Stamenova et al. 2008). The assay is based on

reduction of the tetrazolium dye XTT (2, 3-bis (2-methoxy-

nitro-5 sulfophenyl)-5-[(phenyl-amino)-carbonyl]-2H-tet-

razolium hydroxide). XTT is taken up only by living cells

where it is reduced by O2- to water soluble orange-colored

formazans. A molar extinction coefficient of 2.16 9

104 M-1 S-1 for XTT at 470 nm has been estimated, which

allows determination of the quantity of O2- per alive cell.

The superoxide anions assay was performed immediately

after the treatment with genotoxins and before the cultiva-

tion of cells at 20�C in the retrotransposition test. Results

obtained are presented as pM O2-/cell ± SD.

Materials

Media components were from Difco Chem Co (USA), and

nutritional media were prepared as described (Sherman

et al. 2001). All laboratory genotoxic substances were

purchased from Sigma Ltd. Analytical grade of arsenic

(As) as Na2HAsO4, trivalent chromium (CrIII) as Cr2O3,

hexavalent chromium (CrVI) as CrO3, lead as (CH3COO)2

Pb and zinc as ZnCl2 were used. Genotoxins were dis-

solved in water or dimethylsulfoxide (Me2SO) and dilu-

tions made into the appropriate aqueous treatment

solutions. Me2SO added to the treatment flasks was in no

case more than 5% v/v. S9 fraction from rat liver was

obtained from Microbiological Associates (Rockville,

USA) and S9 mix was prepared as described (Maron

and Ames 1983). N-acetylcysteine was from SIGMA–

ALDRICH, Germany.

Results

Carcinogens generate oxidative stress and activate Ty1

retrotransposition

Previously, a positive Ty1 test response was found for

carcinogens while the noncarcinogenic mutagens did not

activate Ty1 retrotransposition despite the similarity in

chemical structure of the two kinds of genotoxins (Pesheva

et al. 2005, 2008). Here, we repeated this observation and

further extended it by simultaneous measurements of ROS

in tester cells. We used an assay for quantitative estimation

of superoxide anions (O2–) in living cells (Stamenova et al.

2008) and determined the amount of the first synthesized

ROS at the end of the treatment period with the genotoxin.

The carcinogenic or mutagenic status of all compounds and

metals used in our study was according to YARC (1990)

and recent publications in the field (Donkin et al. 2000; Hu

2002). Considerable evidence obtained recently (reviewed

in Bernstein et al. 2005) supports the view that the free bile

acids, such as chenodeoxycholic and lithocholic acids, are

carcinogens in humans, while the conjugated taurodeoxy-

cholic and glycodeoxycholic bile acids did not have car-

cinogenic properties. The results summarized on Table 1

show that all tested carcinogens increased both, the level of

O2– and the rate of Ty1 retrotransposition. The increase

was specific for active carcinogens since procarcinogens

such as benzo(a)pyrene [B(a)P] and benzo(a)antracen

[B(a)A] did not change O2– level and Ty1 retrotransposi-

tion frequency without metabolic activation. The carcino-

gens were tested at concentrations leading to about 50%

survival of cells, determined in preliminary concentration-

dependent experiments. At these similar survival rates, the

different carcinogens generated about fivefold higher level

of O2– with ethylmethanesulfonate (EMS) and the bile

chenodeoxycholic acid (CDH), being the strongest ROS

generators. Opposite to these results, all tested noncarci-

nogenic mutagens did not increase O2– level and Ty1 ret-

rotransposition rate.

The obtained results strongly suggested a relationship

between O2– level and Ty1 retrotransposition frequency in

S. cerevisiae cells treated with carcinogenic genotoxins.

Carcinogenic metals induce ROS production and Ty1

retrotransposition

Some metals (the so-called trace elements) are essential for

the survival of all life forms. Other metals, however, can be

quite genotoxic and carcinogenic. We studied representa-

tives of the two groups with the Ty1 test.

Arsenic (As) and hexavalent chromium (CrVI) are

classified as confirmed human carcinogens, whereas lead

(Pb) has been categorized as probable human carcinogen.

The results obtained with the Ty1 test (Table 2) show that

As and CrVI are strong inducers of Ty1 retrotransposition

with a fold increase of 30 and 70, respectively. As com-

parison, the effect of ethylmethanesulfonate, a well-known

carcinogen, is not so strong, and treatment of tester cells

with equitoxic doses at 50% viability gave a fold increase

for Ty1 retrotransposition of 17 for EMS compared to 27

for As and 46 for CrVI (Tables 1, 2). The toxic heavy

metal Pb gives a moderate increase in the frequency of Ty1

retrotransposition. Its probable carcinogenic status was

based on experiments with laboratory animals evidencing

development of tumors after treatment with lead acetate

(Donkin et al. 2000). Given the high genotoxicity of Pb,

especially for children, it was recommended the carcino-

genic effect of lead to be evaluated in different test

systems. In the Ty1 test, Pb gives a positive response (fold

Arch Toxicol (2011) 85:67–74 69

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increase [2.0), in concentration-dependent manner as did

all tested till now human carcinogens. The data on Table 2

show that the positive responses of Ty1 test to As, CrVI

and Pb appeared at low concentrations having negligible

killing effect and increased with increasing the dose.

Results obtained in kinetics experiments show that with

increasing the time of exposure, the tester cells responded

with increase in the rate of Ty1 retrotransposition, reaching

saturation levels at longer periods of treatments (data not

shown).

Contrary to these results, the treatment with CrIII or Zn

did not enhance the rate of Ty1 retrotransposition, and

background values were obtained even at high concentra-

tions (Table 2) or long exposure. Zinc has been categorized

as not classifiable with regard to human carcinogenicity,

while the data for CrIII carcinogenicity are controversial

(see ‘‘Discussion’’). Although some cells are not permeable

to CrIII, the uptake of chromium through the zinc transport

system was recently demonstrated in S. cerevisiae (Jianglong

et al. 2003; Gitan et al. 2003). The uptake of CrIII in our

experiments was evident by the decreased cell survival

(Table 2), which was similar to the survival rates for cells

treated with CrVI. Therefore, the opposite responses of Ty1

test to CrVI and CrIII are not due to low permeability of

tester cells to CrIII and most likely reflect the property of

the Ty1 test to respond positively only to carcinogens.

The measurements of O2– (Table 3) showed that treat-

ment with the carcinogenic As, CrVI and Pb generated high

levels of ROS and induced Ty1 mobility, as it was found

with other laboratory carcinogenic genotoxins (Table 1).

The induction of Ty1 retrotransposition seems to depend

on the accumulation of O2– since the addition of N-acetyl-

cysteine (NAC), a scavenger of O2–, resulted in loss of both

increased amount of O2– and mobility of Ty1. The treat-

ment with NAC was for 30 min and preceded the addition

of the carcinogen to the culture. As shown on Table 3

(Controls), such treatment was enough to scavenge the

existing O2– in the cells and to not allow an increase in

ROS following the treatment with carcinogen. The results

obtained with CrIII and Zn showed that these Ty1 test

negative metals did not increase O2– level and a pretreat-

ment with NAC is without effect on the rate of Ty1

Table 1 Carcinogens generate high level of O2– and activate Ty1 transposition

Carcinogen

or mutagen

Concentration S9 Mix Survivala (%) O2– (pM/cell)a Ty1 transposition

(fold increase)a

Controls

H2O – - 100 0.58 ± 0.05 1.0

? 98 0.64 ± 0.09 1.1

Me2SO 5% - 96 0.61 ± 0.10 1.1

5% ? 99 0.60 ± 0.12 0.9

Carcinogens

EMSb 16 mM - 54 3.05 ± 0.18 17.5

FAb 40 lg/ml - 48 2.40 ± 0.15 18.1

B(a)P 160 lg/ml - 78 0.63 ± 0.18 1.6

160 lg/ml ? 46 2.85 ± 0.20 58.3

B(a)A 600 lg/ml - 93 0.58 ± 0.08 2.0

600 lg/ml ? 57 2.45 ± 0.16 14.5

CDH 1,000 mM - 53 4.12 ± 0.35 16.0

LH 1,000 mM - 48 2.44 ± 0.19 22.0

Noncarcinogenic mutagens

B(e)P 160 lg/ml - 80 0.66 ± 0.03 1.3

160 lg/ml ? 61 0.68 ± 0.03 1.9

B(b)A 600 lg/ml - 91 0.62 ± 0.05 1.6

600 lg/ml ? 70 0.68 ± 0.03 1.8

A 600 lg/ml - 98 0.48 ± 0.02 1.7

600 lg/ml ? 72 0.80 ± 0.06 1.2

TDH 1,000 mM - 61 0.45 ± 0.04 2.0

GDH 1,000 mM - 80 0.58 ± 0.06 1.8

a Mean values of 6 experiments

Me2SO dimethylsulfoxide, EMS ethylmethanesulfonate, FA formamide, B(a)P benzo(a)pyrene, B(a)A benzo(a)antracene, A anthracene, CDHchenodeoxycholic acid, LH lithocholic acid, TDH taurodeoxycholic acid, GDH glycodeoxycholic acidb EMS and FA were dissolved in water; all other genotoxins were dissolved in Me2SO

70 Arch Toxicol (2011) 85:67–74

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retrotransposition. The effect of ROS scavengers on Ty1

retrotransposition was also studied in experiments with the

carcinogenic genotoxins shown on Table 1. All tested

carcinogens that give positive Ty1 test because of

increased ROS level fail to induce Ty1 retrotransposition in

presence of ROS scavengers (data not shown). These

results evidence a causal relationship between O2– level

and frequency of Ty1 retrotransposition and suggest a key

role of increased ROS level in the activation of Ty1

mobility.

Increased levels of H2O2 also activate Ty1

retrotransposition

Saccharomyces cerevisiae has been shown to have distinct

protective oxidative stress responses to superoxides and

peroxides (Jamieson 1992). A system based mainly on

superoxide dismutases neutralizes increased O2– level. On

the other hand, the YAP1 gene encodes a transcription

activator (Yap1p) which binds to promoters of the target

genes involved in the major defense response against H2O2

and other peroxides (Nguyen et al. 2001). Mutants deleted

for YAP1 accumulate H2O2 intracellularly due to the

absence of detoxifying system (Stephan et al. 1995). We

took advantage from this observation and studied the role

of another ROS member—H2O2 for the carcinogen-

induced Ty1 retrotransposition. Tester cells with disrupted

YAP1 gene (551yap1D) were treated with EMS or CrVI

and the obtained Ty1 retrotransposition frequencies were

compared to the mobility of Ty1 in 551 cells with func-

tioning YAP1 gene (Table 4). The disruption of YAP1 gene

significantly increases Ty1 retrotransposition in response to

Table 2 Concentration

dependence of Ty1 test to

metals

a Actual number of colonies is

given in parenthesisb Average ± SD from 6

experiments

Metal Concentration

(mM)

Survivala

(%)

Ty1 transposants

per 108 survivalbTy1 transposition

(fold increase)

Control (H2O) - 100 (525) 22 ± 2 1.0

As 0.2 83 (434) 90 ± 7 5.0

0.5 62 (326) 362 ± 14 26.5

1.0 49 (245) 305 ± 20 29.7

CrVI 1.0 85 (446) 134 ± 15 7.2

5.0 59 (310) 593 ± 30 45.7

10.0 33 (173) 561 ± 40 77.4

Pb 5.0 80 (420) 47 ± 4 2.7

10.0 50 (263) 57 ± 2 5.2

20.0 12 (63) 22 ± 5 8.3

CrIII 1.0 90 (473) 26 ± 6 1.3

5.0 65 (340) 24 ± 8 1.7

10.0 41 (215) 18 ± 7 2.0

Zn 10.0 72 (378) 28 ± 5 1.8

20.0 68 (357) 31 ± 5 2.0

50.0 66 (346) 24 ± 4 1.7

Table 3 Carcinogenic metals

increase O2– and Ty1

transposition levels

a Average values from 6

experiments

Metal Concentration

(mM)

NAC

(60 mM)

Survivala

(%)

O2– (pM/cell)a Ty1 transposition

(fold increase)a

Control (H2O) - 100 0.64 ± 0.05 1.0

? 91 0.22 ± 0.02 0.9

As 1.0 - 51 3.71 ± 0.55 32.5

1.0 ? 76 0.23 ± 0.03 0.9

CrVI 5.0 - 54 4.40 ± 0.40 47.3

5.0 ? 80 0.41 ± 0.09 1.5

Pb 10.0 - 47 2.83 ± 0.32 18.0

10.0 ? 67 0.13 ± 0.02 1.3

CrIII 5.0 - 54 0.31 ± 0.03 1.3

5.0 ? 61 0.20 ± 0.01 1.1

Zn 50.0 - 63 0.32 ± 0.43 1.7

50.0 ? 75 0.19 ± 0.04 0.9

Arch Toxicol (2011) 85:67–74 71

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treatment with EMS or CrVI, suggesting that carcinogen-

induced Ty1 retrotransposition is activated by elevated

H2O2 levels in the cells. The treatment of 551yap1D cells

with either CrIII or Zn did not affect Ty1 mobility and

values similar to the corresponding controls were found.

The results obtained evidenced that in addition to O2–,

increased H2O2 levels in the cells are also involved in

modulation of Ty1 retrotransposition rates in response to

treatment with carcinogenic genotoxins. This data suggest

that the activation of Ty1 retrotransposition is due to the

general increase in all kinds of ROS. The obtained results

also confirmed the role of increased ROS level in the

activation of carcinogen-induced Ty1 retrotransposition

and indicated that the most likely reason for the selective

positive answer of the Ty1 test to carcinogens is the

increase of ROS level induced by genotoxins.

Discussion

The results obtained in this study provide evidence for a

dependence of carcinogen-induced Ty1 retrotransposition

on increased production of ROS. The Ty1 mobility and O2–

level were studied after treatment with different genotoxins

including laboratory carcinogens, bile acids and metals.

Genotoxins classified as human carcinogens, such as EMS,

B(a)P, B(a)A, the free bile acids CDH, LH and the heavy

metals As, CrVI and Pb were found to generate superoxide

anions and to activate Ty1 retrotransposition. Opposite to

this, genotoxins classified as mutagens without carcino-

genic potential, such as B(e)P, B(b)A, A, the conjugated

bile acids TDH, GDH and the metal Zn did not activate O2–

production and Ty1 retrotransposition. The literature data

for CrIII, which was also studied, are controversial. An

extensive review (Eastmond et al. 2008) points to many

instances of conflicting information. Thus, in vitro data

suggest that CrIII has the potential to react with DNA and

to cause DNA damages which, however, can never occur in

cells under normal circumstances. In vivo evidence sug-

gests that genotoxic effects did not occur in human cells or

animals exposed to CrIII, and the trivalent chromium

complexes are widely used for decades as nutritional sup-

plements and insulin enhancers in patients with type 2

diabetics. Although firm data for a carcinogenic effect of

CrIII are lacking, there is a growing concern (Dillon et al.

2000; Levina and Lay 2008) over the possible carcinoge-

nicity of CrIII based on the assumption that once in the

cell, part of CrIII can be oxidized to CrVI which is a

confirmed human carcinogen. The response of S. cerevisiae

to CrIII was also studied (Jianlong et al. 2003), and the

results obtained show only a moderate inhibition of cell-

growth without perturbation of protein and nucleic acid

synthesis. Contrary to the oxidative stress induced by CrVI,

the trivalent chromium had no effect on ROS balance in

osteoblasts (Fu et al. 2008) and produces a negligible ROS

induction in Hep-2 cells only after chronic treatments for

months (Rudolf and Cervinka 2003). Our results showed

that opposite to CrVI, which induced a burst of ROS and

strongly activated Ty1 retrotransposition, CrIII similarly to

the other studied noncarcinogenic genotoxins, did not gen-

erate oxidative stress in S. cerevisiae and fail to increase Ty1

retrotransposition rate.

The induction of oxidative stress by carcinogens and the

absence of an increase of ROS level after treatment with

noncarcinogenic mutagens of S. cerevisiae cells found in

our study fit well with published data for different cells.

Thus, elevated ROS levels were found after treatment with

EMS of hepatocytes (Tirmenstein et al. 2000; Zhang et al.

2001), with formaldehyde of T lymphocytes (Saito et al.

2005) or whole mice (Jung et al. 2007), with B(a)P and

B(a)A (reviewed in Toyooka and Ibuki 2007), with the

carcinogenic free bile acids DHL and LH (Bernstein et al.

2005), with As (Rodriguez-Gabrial and Russel 2005), CrVI

(Fu et al. 2008) and Pb (Hsu et al. 1997, 1998). Absence of

increase in ROS levels was found after treatment with the

noncarcinogenic B(e)P, B(b)A, anthracene (Toyooka

and Ibuki; 2007; Zbigniev and Wojciech 2006) and Zn

(Sankavarum et al. 2009).

At increased levels, ROS cannot be detoxified suffi-

ciently and may have different deleterious effects, such as

deesterification of phospholipids, accumulation of fatty

acids in membrane bilayers or disturbance of protein

functions (Herrero et al. 2008). ROS also generate a

variety of DNA lesions, including modified bases and

sugars, abasic sites, DNA–protein cross-links, single

strand breaks, chromosome loss (Lawrence and Hinkle

1996; Bohr 2002). Numerous literature data (Salomon

et al. 2004; Tucker and Fields 2004; Lessage and Todeschini

2005; Nyswaner et al. 2008) suggest the role of DNA

Table 4 Ty1 transposition in cells with disrupted YAP1 gene

Strain Genotoxin Survivala

(%)

Ty1 transposition

(fold increase)a

551 - 100 1.0

551 yap1D – 100 1.0

551 EMS (16 mM) 58 14.6

551 yap1D EMS (16 mM) 43 31.6

551 CrVI (5 mM) 51 39.5

551 yap1D CrVI (5 mM) 36 74.1

551 CrIII (5 mM) 62 1.6

551 yap1D CrIII (5 mM) 36 1.8

551 Zn (50 mM) 56 1.5

551 yap1D Zn (50 mM) 48 1.4

a Representative data in one experiment out of three with similar

results

72 Arch Toxicol (2011) 85:67–74

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damages induced by physical and chemical agents in the

activation of Ty1 retrotransposon mobility. Besides direct

DNA damages, the treatment with carcinogenic genotox-

ins also rises secondary DNA damages due to the oxida-

tive stress. Our data fit well with this picture and a likely

explanation of the results obtained would be that in

response to carcinogens both, the direct DNA damaging

effect and the enhanced ROS level play role in the acti-

vation of the Ty1 retrotransposition. It is difficult to assess

which is the first acting event since kinetic experiments

have not yet been performed. Some of our data, however,

suggest a major and independent role of increased ROS

level in the induction by carcinogens of Ty1 mobility.

Thus, when cells were pretreated with N-acetylcysteine to

scavenge the burst of ROS, the treatment with carcinogens

did not induce Ty1 retrotransposition. Quantitative mea-

surements evidenced the absence of ROS induction and

Ty1 activation in these cells, although there is no reason to

suppose that during the time of treatment DNA damages

were not generated by the genotoxin. We suppose that

following the treatment with a carcinogen the cells induce

Ty1 retrotransposition because of increased ROS level and

direct DNA damages. Among the two, the increase of

ROS level seems to be the first acting event and to have

the key role in initiation the activation of Ty1 mobility.

The effect of the direct DNA damages is later multiplied

by the appearance of the secondary DNA damages due to

the already existing oxidative stress. The role of elevated

ROS levels in the induction of Ty1 mobility is also sug-

gested by literature data of other authors (Paquin and

Williamson 1984, 1986) evidencing that cultivation at

suboptimal temperatures of 15–20�C (instead of 30�C)

activates Ty1 retrotransposition without any treatment

with DNA damaging genotoxins. Recently (Zhang et al.

2003), it was found that S. cerevisiae cells generate higher

levels of ROS at 15–20�C compared to cultivation at

30�C.

In conclusion, the results obtained clarified to some

extend the mechanism of the selective positive response

of Ty1 test to carcinogenic genotoxins. The primary and

independent role of increased ROS level for the activa-

tion of Ty1 retromobility found in this study may not be

restricted to the carcinogen-induced Ty1 retrotransposi-

tion only. Previously (Stamenova et al. 2008), it has

been shown that freezing of S. cerevisiae to subzero

temperatures induces Ty1 mobility because of an accu-

mulation of ROS in frozen cells. Further studies are

needed to clarify if increased ROS play also a role in

spontaneous and induced by physical agents Ty1

retrotransposition.

Acknowledgments This research was supported partly by a grant of

the NATO SfP project 977977 given to Pencho Venkov.

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