BioRisk | yf 20 13 | 2 (2022) Apeer-reviewed open-access journal

doi: 10.3897/biorisk.17.77327 RESEARCH ARTICLE & B} te) Re IS k

https://biorisk.pensoft.net

Screening of Amorpha fruticosa and
Ailanthus altissima extracts for genotoxicity/
antigenotoxicity, mutagenicity/antimutagenicity
and carcinogenicity/anticarcinogenicity

Teodora Todorova', Krassimir Boyadzhiev', Aleksandar Shkondrov’,
Petya Parvanova', Maria Dimitrova', Iliana Ionkova’, Ilina Krasteva’,
Ekaterina Kozuharova*, Stephka Chankova'

| Institute of Biodiversity and Ecosystem Research, Bulgarian Academy of Sciences, 2 Gagarin Str, 1113, Sofia,
Bulgaria 2 Department of Pharmacognosy, Faculty of Pharmacy, Medical University of Sofia 2, Dunav Str.,
1000, Sofia, Bulgaria

Corresponding author: Teodora Todorova (tedi_todorova@yahoo.com)

Academic editor: Kalina Danova | Received 1 November 2021 | Accepted 14 December 2021 | Published 21 April 2022

Citation: Todorova T, Boyadzhiev K, Shkondrov A, Parvanova P, Dimitrova M, Ionkova I, Krasteva I, Kozuharova E,
Chankova S (2022) Screening of Amorpha fruticosa and Ailanthus altissima extracts for genotoxicity/antigenotoxicity,
mutagenicity/antimutagenicity and carcinogenicity/anticarcinogenicity. In: Chankova S, Peneva V, Metcheva R,
Beltcheva M, Vassilev K, Radeva G, Danova K (Eds) Current trends of ecology. BioRisk 17: 201-212. https://doi.
org/10.3897/biorisk.17.77327

Abstract

The aim of the present study was to evaluate the potential genotoxic/antigenotoxic, mutagenic/antimuta-
genic, and carcinogenic/anticarcinogenic effect of Amorpha fruticosa (AF) fruit, Ailanthus altissima bark
hexane (AAEH) and methanol (AAEM) extracts on a model system Saccharomyces cerevisiae.

Plants were identified and extracted by Ekaterina Kozuharova. Three concentrations of each extract were tested
— 10, 100 and 1000 pg/ml. In vitro pro-oxidant/antioxidant activities were evaluated by DPPH and DNA to-
pology assay. The potential genotoxic/antigenotoxic, mutagenic/antimutagenic and carcinogenic/anticarcino-
genic effects were revealed in vivo by: Zimmermman’s test on Saccharomyces cerevisiae diploid strain D7ts1,
and Ty retrotransposition test on S. cerevisiae haploid strain 551. Zeocin was used as a positive control.
Based on the in vitro antioxidant activity the extracts could be arranged as follows: AF>AAEM>AAEH.
AAEH possessed moderate oxidative potential. No genotoxic and mutagenic capacity was obtained in
vivo for extracts tested. The levels of total aberrants, convertants and revertants were comparable with the
control ones. No Ty] retrotransposition was induced by extracts treatment. Further, the extracts possessed
well-expressed antigenotoxic, antimutagenic and anticarcinogenic activity. Significant reduction of the
total aberrants, reverse point mutations and Ty1 retrotransposition was obtained. Only the AF extract was

found to reduce the levels of zeocin-induced mitotic gene conversion.

Copyright Teodora Todorova et al. This is an open access article distributed under the terms of the Creative Commons Attribution License (CC
BY 4.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.

202 Teodora Todorova et al. / BioRisk 17: 201-212 (2022)

The three extracts did not possess any genotoxic, mutagenic and carcinogenic effect on Saccharo-
myces cerevisiae. Based on their protective activity, they can be arranged as follows: AF>AAEM>AAEH
which corresponds well with their phytochemical composition. Further experiments could provide more

detailed information concerning the mode of action of extracts, as well as their main constituents.

Keywords
Ailanthus altissima, Amorpha fruticose, carcinogenic/anticarcinogenic, genotoxicity/antigenotoxicity, mu-

tagenic/antimutagenic effect

Introduction

Invasive plant species are considered to be one of the main reasons for biodiversity loss
(Luis et al. 2012; Weidlich et al. 2020; Dyderski and Jagodziriski 2021). Their distri-
bution to new areas has led to massive extinction of plant and animal species in the
last few years (Panjkovié et al. 2021; Szumaniska et al. 2021). Two alien plant species
posing an increasing threat in Bulgaria are Amorpha fruticosa and Ailanthus altissima.
Both are characterized by high tolerance to various habitat conditions and aggressive
invasion due to the lack of suitable herbivores to control their populations (DAISIE
2009; Monaco 2014; Global Invasive Species Database 2019).

A. fruticosa L. (Fabaceae), known as false indigo, false indigo-bush, and bastard
indigobush is a shrub native to North America (Wilbur 1975; USDA NRCS 2009).
The plant has a high quantity of isoflavonoids, rotenoids and prenylated stilbenoids.
Among the prenylated stilbenoids, the group of amorfrutins is quite diverse (Kozu-
harova et al. 2017).

A, altissima (Mill.) Swingle (Simaroubaceae), known as the tree of heaven is na-
tive in China. It was introduced in Europe and North America around the end of the
18" century (Luis et al. 2012; Andonova et al. 2021). It contains alkaloids, terpenoids
and aliphatic volatiles (Kundu and Laskar 2010). The phytochemical composition is
reviewed by Kozuharova et al. (2014). The phytochemical analysis of the bark reveals
the presence of more than 221 compounds such as alkaloids, quassinoids, phenylpro-
panoids, triterpenoids, volatile oils, and other compounds (Li et al. 2021).

Both plants are used in traditional medicine. A. altissima is often applied for the
treatment of asthma, epilepsy, spermatorrhea, bleeding, ascariasis, cold, gastric (dys-
entery) and ophthalmic diseases, etc. (Luis et al. 2012; Kozuharova et al. 2020; Li et
al. 2021). The ethnobotanical application of A. fruticosa is related to the treatment
of stomach pain, intestinal worms, eczema, neuralgia, and rheumatism (discussed in
Kozuharova et al. 2017).

It the present work we hypothesized that A. fruticosa fruit extract and A. altissima
bark extract would be safe and could decrease the zeocin-induced mutagenic, recombi-
nogenic and carcinogenic effects on Saccharomyces cerevisiae model organism. As these
plant species are very invasive growing almost unrestrictedly, they can provide abun-
dant and cheap resources of bioactive compounds. Their pharmacological application

Genotoxicity of Amorpha fruticosa and Ailanthus altissima extracts 203

may lead to excessive harvesting and thus, a decrease in their populations as a strategy
for the protection of native plant habitats. Both plants are promising candidates for the
pharmacology. Even though, data in literature point out that the toxicity evaluation of
the plant extracts is scarce (Kozuharova et al. 2017; Li et al. 2021).

Thus, the aim of the present study was to evaluate the potential genotoxic/antigen-
otoxic, mutagenic/antimutagenic and carcinogenic/anticarcinogenic effect of A. fruti-
cosa fruit extract (AF) and A. altissima bark hexane (AAEH) and methanol (AAEM)

extracts on Saccharomyces cerevisiae.

Materials and methods

Fruits of Amorpha fruticosa were collected in October 2018 from a location near Pasarel
village, Sofia district. Stem bark of Ajlanthus altissima was collected in September 2018
from a location in Sofia, Bulgaria. The plant materials were dried at room temperature,
then pulverized and sieved. The fruits of A. fruticosa were macerated with chloroform
to remove the lipophilic compounds (both the fixed and the essential oils), and then
the material was dried and extracted by percolation with 70% methanol. The solvent
was evaporated on a rotary evaporator; then the extract was lyophilized and named
AE. The stem bark of A. altissima was macerated with hexane to produce the lipophilic
extract, which was dried in vacuo and named AAEH. ‘The resulting defatted substance
was percolated with 70% methanol to obtain the hydrophilic extract, which was con-

centrated, lyophilized and named AAEM.

DPPH radical scavenging activity

The DPPH assay, based on a color reduction of DPPH hydrate from purple to yellow,
was applied as described in Todorova et al. (2015). The radical scavenging activity is
presented as concentration inhibiting 50% of the DPPH radicals. Ascorbic acid was
used as a standard.

DNA topology assay

DNA topology assay was applied according to Todorova et al. (2015). The transforma-
tion of supercoiled pBR322 DNA to a relaxed circular form was photographed with
UV transillumination using G:BOX (Syngene). The relative quantity of supercoiled
DNA was calculated using Image] software.

Treatment of Saccharomyces cerevisiae cells

Stock solutions of A. altissima hexane (AAEH) and methanol (AAEM) extracts dissolved
in 0.1% Tween 20 and A. fruticosa (AF) extract dissolved in sterile MQ water were pre-
pared prior to the experiments. Cell suspensions (1x10’ cells/ml) to the end of the ex-

204 Teodora Todorova et al. / BioRisk 17: 201-212 (2022)

ponential and the beginning of stationary growth phase were pre-treated with three con-
centrations — 10, 100 and 1000 pg/ml of AAEH, AAEM and AF extracts for 30 min at
optimal conditions (30 °C, 200 rpm). Cells were then washed and after that treated with
100 ug/ml Zeocin for 1 min. Single treatment with Zeocin was used as a positive control.
After these procedures, cells were harvested, washed and prepared for further work.

Mutagenicity/antimutagenicity test

Zimmerman’s test was applied on Saccharomyces cerevisiae strain D7ts1 as described in
Todorova et al. (2015); Todorova et al. (2017). The following endpoints were evalu-
ated: cell survival for genotoxic/antigenotoxic and mitotic gene conversion, reverse
mutations and mitotic crossingover — for mutagenic/antimutagenic effects.

Carcinogenicity/anticarcinogenicity test

The Ty1 retrotransposition test applied for iz vivo detection of carcinogenic effect was
used as described by Pesheva et al. (2005) using S. cerevisiae strain 551 as a tester strain.
A “fold increase” higher than two compared to the control, is considered as a positive
response of the Ty1 transposition test.

Statistical analysis

The statistical analysis includes an application of One-way ANOVA with Bonferroni's
post hoc test. P<0.05 was accepted as the lowest level of statistical significance. Concen-
trations inducing 50% inhibition of the cell growth (IC,, values) were calculated using
non-linear regression analysis (GraphPad Prizm5 Software).

Results

Preliminary chemical analysis reveals differences among the chemical composition of
the extracts: AF fruit extract is rich of flavonoids and stilbenoids (amorfrutins A and
B) as it was described previously by (Kozuharova et al. 2017); flavonoids are typical for
the extract of AAEM and terpenoids (sterols) for AAEH that corresponds well to the
data already published by us (Kozuharova et al. 2014).

Antioxidant potential

Slight to moderate radical scavenging activity of the extracts in comparison with the
standard control ascorbic acid was obtained. Based on DPPH assay (Fig. 1), the AF pos-
sesses the best radical scavenging activity calculated as IC,,=63.71 g/mL, followed by the
AAEM with IC,,=696.12 g/mL. The AAEH show the lowest radical scavenging poten-
tial IC,,= 1396.97 g/mL). The IC,, of the ascorbic acid was calculated as 15.94 g/mL.

Genotoxicity of Amorpha fruticosa and Ailanthus altissima extracts 205

100

2
2
5 80
> 60
a
ey

~ 4
g 2 40 _s
0
r
i
~
%
wo 0 200 400 600 800 4000 4200

Extract concentration (pg/ml)
——AF -—a-AAEM .--s--AAEH

Figure |. Radical scavenging activity (%) of Amorpha fruticosa, Ailanthus altissima methanolic and hex-

ane extract. Data are presented mean values from at least three independent experiments.

To evaluate the oxidative potential of the extracts DNA topology assay was performed.
This assay provides information not only about the oxidative/antioxidant but also on the
DNA damaging/protective effect of the tested extracts. Based on the calculated relative
quantity of supercoiled DNA the extracts could be arranged as follows: AF>AAEM>AAEH
(Fig. 2). The hexane extract was shown to possess moderate oxidative potential.

Comparing antioxidant properties of the extracts, the moderate protection of
AAEH was detected depending on the concentration — 500 and 1000 ug/mL. AF and
AAEM did not show good antioxidant properties towards the hydroxyl anions (Fig. 3).

he
—
ee
tw
=

ww eR eS =
ecco =

@AF
O AAEM
0 AAEH

i

control Fe2+ 10 100-500: 1000
ions Extract concentration (g/ml)

aoG

Relative quantity of
supercoiled DNA (%)

Figure 2. Agarose gel electrophoresis for studying possible DNA damaging effect. Agarose gel electro-
phoretic patterns of plasmid DNA treated with A. fruticosa L. A Ailanthus altissima methanolic B and
hexane C extract in the absence of Fe** ions (0.08 mM): lane 1 — DNA control; lane 2 — Fe** ions control;
lane 3 — 10 pg/mL extract; lane 4— 100 pg/mL extract; lane 5 — 500 pg/mL extract; lane 6 — 1000 pg/mL

extract D Densitometrical estimation of the relative quantity of supercoiled DNA.

206 Teodora Todorova et al. / BioRisk 17: 201-212 (2022)

$3

@AF

_ WN) EM A es

control Fe2+ 10 100 500 1000
ions

—
=

Relative quantity of
supercoiled DNA (%o)
S ses

Extract concentration (ug/ml)

Figure 3. Agarose gel electrophoresis for studying possible DNA protective effect against Fe** ions.
Agarose gel electrophoretic patterns of plasmid DNA treated with A. fruticosa L. A Ailanthus altissima
methanolic B and hexane C extract in the presence of Fe** ions (0.08 mM): lane 1 - DNA control; lane
2 — Fe* ions control; lane 3 — Fe** ions and 10 pg/mL extract; lane 4 — Fe* ions and 100 ug/mL extract;
lane 5 — Fe* ions and 500 pg/mL extract; lane 6 - Fe** ions and 1000 pg/mL extract D Densitometrical
estimation of the relative quantity of supercoiled DNA.

Mutagenicity/antimutagenicity

The survival after the treatments was comparable with the negative control — untreated
cells. No effect was obtained regarding the genetic events — convertant and revertant
frequencies as well as total aberrants (Table 1). The three extracts did not possess geno-
toxic and mutagenic properties at the studied concentrations.

Concerning the antimutagenic properties, an increase in the cell survival in
comparison with the positive control was measured after all the treatments. Sig-
nificant reduction of the reverse mutations to levels comparable with that in un-

Table |. Frequency of gene conversion in ¢rp5 locus, reversion in i/v1-92 allele and mitotic crossing-over
in ade2 locus after single treatment of S. cerevisiae D7ts1 with 10, 100 and 1000 pg/ml AK AAEM and
AAEH. Zeocin was used as a positive control.

Extract concentration Zeocin (ug/ml) Survival (%) Gene conversion/ Reversion/ 10° cells Total aberrants (%)
(ug/ml) 10° cells

0 0 100 1.02+0.01 0.003+0.0002 0.4374£0.021***

0 100 32.9972 750 4.3040.7*** 0.038+0.0005*** 2.3314£0.667***

10 0 99.62+1.21*** 1.05+0.05*** 0.002+0.00009*** = -0.576+0.150***

= 100 0 99.054+3.93*** 1.07+0.01*** 0.002+0.0001*** 0.74140.163***
1000 0 99.97+1.64*** 1.07+0.07*** 0.00140.00004*** = 0.507+40.013***

S 10 0 99.4142.13*** 1.15+0.05*** 0.002+0.00005*** 0.498 +0.130***
= 100 0 99.374+3.01*** 1.13+0.03*** 0.003+0.00005*** = -0.501+0.021***
1000 0 98.3141.59*** 1.16£0.07*** 0.002+0.00009*** 0.542+0.043**

es 10 0 96.47£1.98*** 1.90+0.08*** 0.004+0.00006*** 0.602+0.046**
= 100 0 89.1142.04*** 1.60£0.05*** 0.004+0.00002*** 0.689+0.016**
1000 0 97.1442.43*** 2.02+0.04*** 0.006+0.00001*** 0.802+0.112**

Frequencies are means + SEM, n=4. The significance of differences between positive control (Zeo) and treatment with various extracts’
concentrations were calculated by ANOVA with a post-hoc test Bonferroni’s Multiple Comparison Test (**P<0.01; ***P < 0.001).

Genotoxicity of Amorpha fruticosa and Ailanthus altissima extracts

207

Table 2. Frequency of gene conversion in trp5 locus, reversion in i/v1-92 allele and mitotic crossing-over
in ade2 locus after pre-treatment of S. cerevisiae D7ts1 with 10, 100 and 1000 ug/ml AF, AAEM or AAEH
followed by treatment with 100 pg/ml Zeocin.

Extract concentration Zeocin (ug/ml)

Survival (%)

Gene conversion/ Reversion/ 10° cells Total aberrants (%)

(ug/ml) 10° cells
0 0 100 0.52+0.01 0.003+0.0002 0.437+0.021
0 100 32.99+2.75*** 4.30+0.70*** 0.038+0.0005*** 2.331£0.667***
10 100 94.934+4.08*** 0.75£0.50 *** 0.005+0.0009*** 0.56+0.16**
= 100 100 77 5S441.51°** 0.81+0.10 *** 0.006+0.0002*** 0.74£0.13**
1000 100 71.01£9.07°°* 0.76+0.09 *** 0.005+0.0004*** 0.50£0.03**
S 10 100 87.68+1.46*** 2.34+0.84 " 0.021+0.0006*** 1.51+0.035 *°
= 100 100 76.83+2.41*** 3.20+0.57 0.005+0.00014*** 0.57£0.056 **
1000 100 58.06+2.11*** 2.84+£0.39 "s 0.006+0.00057*** 0.76£0.03 ™*
Rs 10 100 77.71£3.65°™* 6.9340.79* 0.014+0.0035*** 1.13£0.078 *
= 100 100 FiDatAS oT 3.89£0.53 " 0.0314£0.0046 * 0.92+0.09 **
1000 100 84.6041.24*** 2.90+£0.67 "* 0.032+0.0086 ™ 0.8540.05 ™*

Frequencies are means + SEM, n=4. The significance of differences between positive control (Zeo) and treatment with various extracts’
concentrations was calculated by ANOVA with a post-hoc test Bonferroni’s Multiple Comparison Test (NS: nonsignificant; *P<0.05;
(**P<0.01; ***P < 0.001).

treated control was obtained after the pre-treatment with AF and AAEM with-
out concentration’s effect. Around 2.5-fold lower levels were measured after pre-
treatment with 10 pg/ml AAEH (Table 2). AF at all the concentrations decreased
the zeocin-induced mitotic gene conversion. No effect on this genetic event was
obtained after pre-treatment with AAEM, while a potentiation of the zeocin re-
combinogenicity was observed after pretreatment with 10 ug/ml AAEH. The per-
cent of total aberrants after the pre-treatments was also lower than that measured
after single zeocin treatment.

Carcinogenic/anticarcinogenic potential

Data revealed that single treatment with 1000 g/ml AF possesses slight dose-depend-
ent genotoxic effect, reducing the cell survival of strain 551 to 78% (Fig. 4A). None
of the other extracts affected the cell survival. On the other side, pre-treatment results
in around 2-fold increased cell survival for all the concentrations of the extract in
comparison with the cell survival after single zeocin treatment (Fig. 4A). No dose-
dependent enhancement of cell survival is observed. The Ty1 retrotransposition events
are also found. Our results clearly indicate that none of the tested concentrations of AF,
AAEM and AAFH can induce Ty] retrotransposition in Saccharomyces cerevisiae. These
data suggest no carcinogenic properties of the extracts.

Further, well expressed anti-carcinogenic activity, measured as a reduction of the
transposition rate to levels comparable with the negative control is defined when pre-
treatment with concentrations of AF and AAEH is applied (Fig. 4B). The only con-
centration that cannot protect cells from damaging action of Zeocin is the lowest con-

centration of AAEM — 10 pg/ml.

208 Teodora Todorova et al. / BioRisk 17: 201-212 (2022)

A) © without Zeo B) # 4
150 EI with Zeo =
‘ |
= a
. : 2
F 4 a
= i]
= 5?
o 2
ed 2 ] ° ‘ ; fs *
5 Po FF ES ES = Smal:
100 1000) 10 100 1000

AF AAEM AAEH AF AAEM
(ug/ml) (ug/ml) (ug/ml) (ug/ml) (ug/ml) (ng/ml)
Treatment Treatment

Figure 4. Cell survival A and Ty1 retrotransposition rates B of Saccharomyces cerevisiae strain 551 after
treatment with 10, 100 and 1000 pg/ml AK AAEM and AAEH with or without Zeocin. Where no error
bars are evident, errors are equal or less than the symbols.

Discussion

Slight to moderate antioxidant activity in vitro of extracts is identified. Such activity of
Amorpha fruticosa does not correspond to data published by Zheleva-Dimitrova (2013)
and Ivanescu et al. (2019). The variation in the radical scavenging activity could be
explained by different phytochemical composition based on the geographical origin
of plant, variations in the plant extraction and the methodology, etc. Consistence be-
tween DNA topology assay results and those obtained by DPPH is found. Moderate
oxidative potential leading to single-strand pDNA damage is found for hexane extract.

Our in vivo experiments were performed on Saccharomyces cerevisiae. Saccharomyces
cerevisiae has been chosen as a model system for human cell due to the similarities in
main stress response pathways (discussed in Todorova et al. 2015). Moreover, experi-
ments on yeasts could be a valuable tool when taking into consideration the Directive
2010/63/EU. This directive is aiming to anchor firmly the ,,Principle of the Three Rs”
— To Replace, Reduce and Refine” the use of animals for experimental and scientific
purpose in the EU Member States. According to the Annex (47) there is a need to
develop new methods alternative to animal testing and proposed to validation in the
European Union Reference Laboratory for alternatives to animal testing (EURL EC-
VAM) (http://ihcp.jrc.ec.europa.eu/our_labs/eurl-ecvam). Additionally, experiments
on diploid and haploid yeast cells allow obtaining new fundamental information con-
cerning the potential lethal effect of various chemical and physical agents and genetic
instability (Evstratova et al. 2018).

The results obtained by us reveal that the three extracts do not affect the cell sur-
vival, the mitotic gene conversion in trp5 locus, reversion in ilv1-92 allele, mitotic
crossing-over in ade2 locus and Ty1 retrotransposition. From our point of knowledge
this is the first finding that Amorpha fruticosa fruit extract and Ailanthus altissima
bark extracts are not genotoxic, mutagenic and carcinogenic in our test-system.

Our research has been extended in order to evaluate the possible antigenotoxic,
antimutagenic and anticarcinogenic potential of the extracts against the action of

Genotoxicity of Amorpha fruticosa and Ailanthus altissima extracts 209

the radiomimetic Zeocin. Zeocin was chosen as a damaging agent due to several
reasons: it is a radiomimetic, member of the bleomycin family of antibiotics, that
damages DNA in a way similar to that of ionizing radiation; possesses pro-oxidative
capacity (Chankova et al. 2013; Todorova et al. 2015), mutagenic, and carcino-
genic effect in Saccharomyces cerevisiae (Todorova et al. 2015), clastogenic, DNA
damaging, and genotoxic effects in microalgae, higher plants, and human lympho-
cyte cell culture (Chankova et al. 2007; Dimova et al. 2009; Kopaskova et al. 2011;
Gateva et al. 2015).

In this study it was demonstrated that pre-treatment with extracts could protect
cells from the genotoxic action of Zeocin measured as cell survival. No relation between
the cell survival and pre-treatment concentrations was identified. Concerning the an-
timutagenic capacity of extracts the specificity of their action was obvious. Significant
reduction of the total aberrants was obtained after the treatment with the extracts. The
only extract reducing the mitotic gene conversion was AF. No effect was observed after
the pre-treatment with AAEM. A significant increase in the levels of this genetic event
was measured when pre-treatment with 10 ug/ml AAEH which is in accordance with
another study where sterols are reported to potentiate the activity of another member
of the zeocin family — bleomycin (Hoffmann et al. 2011). Such differences in the activ-
ity could be related to the phytochemical content of the extracts. Isoflavonoids are the
major constituents of AF extract while AAEH is characterized by the predominance
of phytosterols. Flavonoids are already known to possess good antimutagenic proper-
ties. Based on the available literature and the present results it could be suggested that
AF with flavonoinds as main constituents may protect yeast cells from zeocin-induced
mitotic gene conversion and crossing over by activation of HR repair and modulation
of chromatin structure.

On the other side, significant amelioration of the reverse point mutations and Ty1
retrotransposition was observed. It is well known that the antimutagenic and anticar-
cinogenic properties could be related to significant antioxidant activity or to activation
of DNA repair processes. As in our im vitro experiments evidence was provided for
mild to moderate antioxidant activity of extracts tested, it could be suggested that in
this case the reduction of the genetic events is not related to the antioxidant potential.
Having in mind that the reverse mutation frequency is used for measurement of error
prone recombination (Mitchel and Morrison 1986), we could speculate that the po-
tential mechanism of action of the extracts may be an activation of protective enzymes
independent of those required for HR.

From our point of knowledge for the first time it was shown by us that Amorpha
fruticosa fruit extract and Ailanthus altissima bark extracts possess no genotoxic, muta-
genic and carcinogenic capacity on a model system Saccharomyces cerevisiae.

Based on their protective activity, they can be arranged as follows: AF>~AAEM>AAEH
that corresponds well with their phytochemical composition. Further experiments
could provide more detailed information concerning the mode of action of extracts, as
well as their main constituents.

210 Teodora Todorova et al. / BioRisk 17: 201-212 (2022)

Acknowledgements

This work has been carried out in the framework of the National Science Program “En-
vironmental Protection and Reduction of Risks of Adverse Events and Natural Disas-
ters’, approved by the Resolution of the Council of Ministers N° 577/17.08.2018 and
supported by the Ministry of Education and Science (MES) of Bulgaria (Agreement
N° JJ01-363/17.12.2020).

References

Andonova TG, Dimitrova-Dyulgerova IZ, Slavov Z, Dincheva IN, Stoyanova AS (2021) Vola-
tile compounds in flowers, samaras, leaves and stem bark of Ailanthus altissima (Mill.)
Swingle, growing in Bulgaria. IOP Conference Series: Materials Science and Engineering
1031(1): e012087. https://doi.org/10.1088/1757-899X/1031/1/012087

Chankova SG, Dimova E, Dimitrova M, Bryant PE (2007) Induction of DNA double-strand
breaks by Zeocin in Chlamydomonas reinhardtii and the role of increased DNA double-
strand breaks rejoining in the formation of an adaptive response. Radiation and Environ-
mental Biophysics 46(4): 409-416. https://doi.org/10.1007/s00411-007-0123-2

Chankova S, Todorova T, Parvanova P, Miteva D, Mitrovska Z, Angelova O, Imreova P, Mu-
caji P (2013) Kaempferol and jatropham: Are they protective or detrimental for Chla-
mydomonas reinhardtii? Dokladi na Bulgarskata Akademia na Naukite 66: 1121-1128.

DAISIE (2009) Handbook of Alien Species in Europe. Dordrecht.

Dimova E, Dimitrova M, Miteva D, Mitrovska Z, Bryant PE, Chankova S (2009) Does single-
dose cell resistance to the radiomimetic Zeocin correlate with a Zeocin-induced adaptive
response in Chlamydomonas reinhardtii strains? Radiation and Environmental Biophysics
48(1): 77-84. https://doi.org/10.1007/s0041 1-008-0199-3

Dyderski MK, Jagodzinski AM (2021) How do invasive trees impact shrub layer diversity
and productivity in temperate forests? Annals of Forest Science 78(1): 1-14. https://doi.
org/10.1007/s13595-021-01033-8

Evstratova ES, Mishra KP, Petin VG (2018) The comparison of genetic instability in haploid
and diploid yeast cells exposed to ionizing radiations of different linear energy transfer
and ultraviolet light. Journal of Radiation and Cancer Research 9(2): 79-85. https://doi.
org/10.4103/jrer.jrcr_6_18

Gateva S, Angelova O, Chankova S (2015) Double-strand breaks detection in human lympho-
cytes by constant field gel electrophoresis. Dokladi na Bulgarskata Akademia na Naukite
68(4): 469-475. https://doi.org/10.1080/09553009314450801

Global Invasive Species Database (2019) Species profile: Ailanthus altissima. http://www.iucng-
isd.org/gisd/species.php?sc=319 [accessed: 25-07-2019]

Hoffmann GR, Laterza AM, Sylvia KE, Tartaglione JP (2011) Potentiation of the mutagenic-
ity and recombinagenicity of bleomycin in yeast by unconventional intercalating agents.
Environmental and Molecular Mutagenesis 52(2): 130-144. https://doi.org/10.1002/
em.20592

Genotoxicity of Amorpha fruticosa and Ailanthus altissima extracts ola

Ivanescu B, Lungu C, Vlase L, Gradinaru AC, Tuchilus C (2019) HPLC analysis of phenolic
compounds, antioxidant and antimicrobial activity of Amorpha fruticosa L. extracts. Jour-
nal of Plant Development 26: 77-84. https://doi.org/10.33628/jpd.2019.26.1.77

Kopaskova M, Hadjo L, Yankulova B, Jovtchev G, Galova E, Sevcovicova A, Mucaji P, Mia-
dokova E, Bryant P, Chankova S (2011) Extract of Lillium candidum L. can modulate
the genotoxicity of the antibiotic Zeocin. Molecules (Basel, Switzerland) 17(1): 80-97.
https://doi.org/10.3390/molecules17010080

Kozuharova E, Lebanova H, Getov I, Benbassat N, Kochmarov V (2014) Ailanthus altissima
(Mill.) Swingle-a terrible invasive pest in Bulgaria or potential useful medicinal plant.
Bothalia journal 44(3): 213-230.

Kozuharova E, Matkowski A, Wozniak D, Simeonova R, Naychov Z, Malainer C, Atanasov AG
(2017) Amorpha fruticosa—A noxious invasive alien plant in Europe or a medicinal plant
against metabolic disease? Frontiers in Pharmacology 8: e333. https://doi.org/10.3389/
fphar.2017.00333

Kozuharova E, Benbassat N, Berkov S, Ionkova I (2020) Ailanthus altissima and Amorpha fru-
ticosa—invasive arboreal alien plants as cheap sources of valuable essential oils. Pharmacia
67(2): 71-81. https://doi.org/10.3897/pharmacia.67.e483 19

Kundu P, Laskar S (2010) A brief resume on the genus Ailanthus: Chemical and pharmacological
aspects. Phytochemistry Reviews 9(3): 379-412. https://doi.org/10.1007/s11101-009-9157-1

Li X, Li Y, Ma S, Zhao Q, Wu J, Duan L, Xie Y, Wang S (2021) Traditional uses, phytochemis-
try, and pharmacology of Ailanthus altissima (Mill.) Swingle bark: A comprehensive review.
Journal of Ethnopharmacology 275: e114121. https://doi.org/10.1016/j.jep.2021.114121

Luis A, Gil N, Amaral ME, Domingues F, Duarte APC (2012) Ailanthus altissima (Miller)
Swingle: A source of bioactive compounds with antioxidant activity. BioResources 7(2):
2105-2120. https://doi.org/10.15376/biores.7.2.2105-2120

Mitchel REJ, Morrison DP (1986) Inducible error-prone repair in yeast suppression by heat
shock. Mutation Research. Fundamental and Molecular Mechanisms of Mutagenesis
159(1-2): 31-39. https://doi.org/10.1016/0027-5107(86)90109-0

Monaco A (2014) European guidelines on protected areas and invasive alien species. Council
of Europe, Rome.

Panjkovié B, Rat M, Mihajlovi¢ S, Galambos L, Kis A, Puzovié S, Nadazdin B, Seat J,
Vukajlovi¢ E, Tot I, Dapi¢ M (2021) Invasive Alien Species in the Balkan Peninsula. Inva-
sive Alien Species: Observations and Issues from Around the World 3: 42-87. https://doi.
org/10.1002/9781119607045.ch27

Pesheva M, Krastanova O, Staleva L, Dentcheva V, Hadzhitodorov M, Venkov P (2005) The
Ty1 transposition assay: a new short-term test for detection of carcinogens. Journal of mi-
crobiological methods 61(1): 1-8. https://doi.org/10.1016/j.mimet.2004.10.001

Szumaniska I, Lubiriska-Mielinska $, Kaminski D, Rutkowski L, Nienartowicz A, Piernik A
(2021) Invasive Plant Species Distribution Is Structured by Soil and Habitat Type in the
City Landscape. Plants 10(4): e773. https://doi.org/10.3390/plants10040773

Todorova T, Pesheva M, Gregan F, Chankova S (2015) Antioxidant, antimutagenic and an-
ticarcinogenic effects of Papaver rhoeas L. extract on Saccharomyces cerevisiae. Journal of

Medicinal Food 18(4): 460-467. https://doi.org/10.1089/jmf.2014.0050

Zi? Teodora Todorova et al. / BioRisk 17: 201-212 (2022)

USDA NRCS (2009) The PLANTS Database (http://plants.usda.gov, 24 June 2009). National
Plant Data Center, Baton Rouge, LA, 70874-4490.

Weidlich EW, Florido FG, Sorrini TB, Brancalion PH (2020) Controlling invasive plant species
in ecological restoration: A global review. Journal of Applied Ecology 57(9): 1806-1817.
https://doi.org/10.1111/1365-2664.13656

Wilbur RL (1975) A revision of the North American genus Amorpha (Leguminosae-Psoraleae).
Rhodora 77(811): 337-409.

Zheleva-Dimitrova DZ (2013) Antioxidant and acetylcholinesterase inhibition properties of
Amorpha fruticosa L. and Phytolacca americana L. Pharmacognosy Magazine 9(34): 109.
https://doi.org/10.4103/0973-1296.111251