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Abstract and Figures

DNA integrity is considered an important parameter of semen quality and is of significant value as a predictor of male fertility. Currently, there are several methods that can assess sperm DNA integrity. One such assay is the comet assay, or single-cell gel electrophoresis, which is a simple, sensitive, reliable, quick and low-cost technique that is used for measuring DNA strand breaks and repair at the level of individual cells. Although the comet assay is usually performed with somatic cells from different organs, the assay has the ability to detect genotoxicity in germ cells at different stages of spermatogenesis. Since the ability of sperm to remove DNA damage differs between the stages, interpretation of the results is dependent on the cells used. In this paper we give an overview on the use and applications of the comet assay on mature sperm and its ability to detect sperm DNA damage in both animals and humans. Overall, it can be concluded that the presence in sperm of significantly damaged DNA, assessed by the comet assay, is related to male infertility and seems to reduce live births. Although there is some evidence that sperm DNA damage also has a long-term impact on offspring’s health, this aspect of DNA damage in sperm is understudied and deserves further attention. In summary, the comet assay can be applied as a useful tool to study effects of genotoxic exposures on sperm DNA integrity in animals and humans.
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7Institute for Medical Research and Occupational Health, Mutagenesis Unit, Zagreb, Croatia
8University of Zagreb, Centre for Research and Knowledge Transfer in Biotechnology, Zagreb, Croatia
9Maastricht University, School for Nutrition and Translational Research in Metabolism (NUTRIM), Department of Pharmacology & Toxicology, Maastricht, the Netherlands
:University of Oslo, Institute of Basic Medical Sciences, Department of Nutrition, Oslo, Norway
;Norwegian Institute for Air Research (NILU), Department of Environmental Chemistry, Health Effects Laboratory, Kjeller, Norway
<Norwegian Institute of Public Health (NIPH), Section of Molecular Toxicology, Department of Environmental Health, Oslo, Norway
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1. Introduction
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UNCORRECTED PROOF
G. Gajski et al. Mutation Research-Reviews in Mutation Research xxx (xxxx) 108398
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Box 1.
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UNCORRECTED PROOF
G. Gajski et al. Mutation Research-Reviews in Mutation Research xxx (xxxx) 108398
3. Techniques for evaluating DNA damage in sperm
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JE IE <7H FK8B?I>;: :7J7 FEI?J?L; 9EDJHEBI 7H; KIK7BBO I;C;D I7CFB;I
JH;7J;: M?J> >O:HE=;D F;HEN?:; 7D: 1H7OI 7BJ>EK=> H;I;7H9>;HI >7L;
7BIE KI;: =BO9?:7C?:; 8;DP;D; 7D: :?;J>OBIJ?B8E;IJHEB , 7I M;BB 7I
'7I;  C7@EH FHE8B;C M?J> J>; IF;HC 9EC;J 7II7O ?I J>; B79A E< 7
=EE: FEI?J?L; 9EDJHEB I?D9; J>; B;L;BI E< ' :7C7=; E8J7?D;: M?J>
IJ7D:7H: ,, ?D:K9?D= 7=;DJI >O:HE=;D F;HEN?:; 7D: 1H7OI :?<<;H
9ECF7H;: JE IEC7J?9 9;BBI FEII?8BO :K; JE J>; L;HO J?=>J FHEJ7C?D;
F79A7=?D= E< IF;HC 45 DEJ>;H 9KHH;DJ FHE8B;C ?I J>; ;N9;II?L; ?D
J;HB78EH7JEHO L7H?78?B?JO 7D: KI; E< C7DO :?<<;H;DJ FHEJE9EBI >;D9;
7JJ;CFJI JE IJ7D:7H:?P; 7D: >7HCED?P; J>;I; FHEJE9EBI I>EKB: 8; KD
:;HJ7A;D JE ;D78B; 7 :?H;9J 9ECF7H?IED E< H;IKBJI 7D: JE ;IJ78B?I> J>;
IF;HC 9EC;J 7II7O 7I 7 L7B?: :?7=DEIJ?9 JEEB 45
5. Modi0ed comet assay protocols for the evaluation of DNA
damage in sperm
->;H; 7H; 7 <;M CEH; L7H?7DJI 7D: CE:?q97J?EDI ?D 7::?J?ED JE J>;
78EL;C;DJ?ED;: IJ;FI
+;)HEEC;J +;F7?H )HEq9?;DJ EC;J ?I 7D in vitro C;J>E: JE 7I
I;II ' :7C7=; ?D C7CC7B?7D IF;HC ->; CE:?q97J?ED ?DJHE:K9;: ?I
87I;: ED J>; 7::?J?ED E< 7 9HK:; FHEJ;?D ;NJH79J <HEC IEC7J?9 9;BBI
D7C;BO <HEC !;%7 9;BBI JE 7=7HEI;;C8;::;: IF;HC ED C?9HEI9EF?9
IB?:;I 45 ->; =;D;H7B ?:;7 E< J>?I 7FFHE79> ?I J>7J ' H;F7?H ;D
POC;I ?D J>; FHEJ;?D ;NJH79J M?BB H;9E=D?P; :7C7=; ?D IF;HC 9;BBI 7D:
9EDL;HJ J>7J :7C7=; ?DJE :;J;9J78B; IJH7D: 8H;7AI "D EH:;H JE J;IJ J>;
FHEJE9EB 8KBB IF;HC M;H; JH;7J;: in vitro M?J> C;J>OB C;J>7D;IKB<ED7J;
&&, EH C;BF>7B7D &%) 7D: J>; 9EC;J 7II7O M7I 9ED:K9J;: 8EJ>
M?J> 7D: M?J>EKJ 7::?J?ED E< 7 FHEJ;?D ;NJH79J .I?D= J>; IJ7D:7H: FHE
JE9EB M?J>EKJ FHEJ;?D IKFFB;C;DJ7J?ED DE ;<<;9J E< &&, EH &%) M7I
:;J;9J;: ED IF;HC ' :7C7=; "D 9EDJH7IJ 7 :EI;:;F;D:;DJ ;<<;9J
M7I E8I;HL;: 7<J;H 7::?J?ED E< J>; FHEJ;?D ;NJH79J FH;IKC78BO H;IKBJ?D=
<HEC ?D9?I?ED E< ' 8O ' H;F7?H ;DPOC;I 7J I?J;I E< &&, EH &)%
7BAOB7J?ED ->?I :;CEDIJH7J;I J>; FEJ;DJ?7B E< J>; FHEJE9EB JE :;J;9J
:7C7=; J>7J ?I KIK7BBO DEJ :;J;9J;: 8O J>; 9EC;J 7II7O
&7DO C;J>E:EBE=?;I JE ;L7BK7J; ' 8H;7A7=; ?D IF;HC7JEPE7 7H;
KD78B; JE :?<<;H;DJ?7J; 8;JM;;D I?D=B;IJH7D:;: ' 8H;7AI 7D: :EK
8B;IJH7D:;: ' 8H;7AI ?D J>; I7C; IF;HC !;D9; 7 JMEJ7?B;: 9EC;J
7II7O -EC;J ;CFBEO?D= JME :?C;DI?ED7B ;B;9JHEF>EH;I?I <EH 7I
I;II?D= ' :7C7=; ?D IF;HC7JEPE7 M7I :;L;BEF;: JE EL;H9EC; J>?I
UNCORRECTED PROOF
G. Gajski et al. Mutation Research-Reviews in Mutation Research xxx (xxxx) 108398
Table 1
->; IF;HC 9EC;J 7II7O <EH J>; ;L7BK7J?ED E< ' :7C7=; ?D ?DL;HJ;8H7J;I
D?C7B -OF;
E<
IJK:O
=;DJIJH;IIEH ED9;DJH7J?ED H7D=; )7H7C;J;HI
J;IJ;:
+;IFEDI; +;<;H;D9;I
Molluscs Scrobicularia
plana
In
vitro
475) 475)  7D:  i=%  J7?B ' 45
Haliotis midae In
vivo
!( CCEB%  J7?B '
(-& " 45
Crassostrea gigas In
vitro
9HOEFH;I;HL7J?ED
9HOEFHEJ;9J7DJI
<H;;P?D=J>7M?D= !% ! !"
-EJ% -EJ
-EJ" -%
- -"
45
In
vitro
>;H8?9?:;I :?KHED =BOF>EI7J;
7D: +EKD:KF !(7I )
    7D:  i=% !(
  7D:  i&
 J7?B ' :?KHED  i=%
g =BOF>EI7J; g
+EKD:KF !(
i&
45
In
vivo
:?KHED -ME :7O ;NFEIKH; FKBI;I 7J  7D:  i=%  J7?B ' 45
Annelids Eisenia fetida In
vitro,
in
vivo
γH7:?7J?ED 1H7OI In vivo E γH7:?7J?ED :EI; H7J;I 
 C O> 1H7OI  O> in vitro 
   EH  O 1H7OI
 J7?B ' 45
In
vivo
HI;D?J;     7D:  C= IA= (-&  C= IA= 45
Arenicola
marina
In
vitro
&&, &&,   7D:  C=%  J7?B ' ↑ ≥  C=% 45
Arthropods Gammarus
fossarum
In
vitro,
in
vivo
&&, ;DL?HEDC;DJ7B
9EDJ7C?D7DJI :B $‚H‚(ƒ
F7H7GK7J &) 7D: 475)
In vitro    7D:  CCEB% in vivo  
7D:  CEB%
 J7?B ' in vitro  CCEB%
in vivo  CEB%
$‚H‚(ƒ F7H7GK7J
&)
45
In
vivo
&&,    7D:  C=%  J7?B '  C=% 45
In
vivo
-;CF;H7JKH; 9ED:K9J?L?JO  J7?B ' g 45
In
vivo,
in
situ
&&, $H( 00-) In vivo &&,   7D:  C=% 7D:
$H(  7D:  C=% in situ
EKH8H; +?L;H 7D: ?ED +?L;H H7D9;
 J7?B ' 45
In
situ
00-) +>`D;BF;I +?L;HI H7D9;  J7?B ' 45
Gammarus
elvirae
In
vivo
!= )8 !=   7D:  μ=% )8   7D:
 μ=%
 J7?B ' !=  i=% )8
 i=%
45
Echinogammarus
veneris
In
vivo
!= )8 !=   7D:  μ=% )8   7D:
 μ=%
 J7?B ' !=  i=% )8
 i=%
45
Macrobrachium
rosenbergii
In
vivo
--   7D:  C=% -%  J7?B
' (-&  C=% 45
Palaemon
serratus
In
vitro
(FJ?C?I7J?ED !( ./
&&,
./   7D:  #C !( 
7D:  μ& &&,   7D:  C&
/, . 45
In
situ
8?EJ?9 <79JEHI M7J;H
J;CF;H7JKH; ;DL?HEDC;DJ7B
FEBBKJ?ED
,;?D; 7O 'EHC7D:O H7D9; /, . 45
In
vivo
"CF79J E< F7J;HD7B :7C7=;:
' ED J>; H;FHE:K9J?L;
IK99;II &&,    7D:
 μ&
&&,    7D:  μ& /, . 45
Palaemon
longirostris
In
situ
)7II?L; 7D: 79J?L; H;9EL;HO E<
M?B: IF;9?C;DI
,;?D; ;IJK7HO 'EHC7D:O H7D9; /, . g I;7IED
:;F;D:;DJ
45
Drosophila
simulans
In
vivo
0EB879>?7?D<;9J?ED +(, /,  J7?B
' 45
Paracentrotus
lividus
In
vivo
3D( ') NFEI;: J>HEK=> J>; :?;J JE :?s;H;DJ I?P;I 
7D:  DC 3D(')I  7D:  C= 3DA=
3D(')I  DC 7D:  7D:  C= 3DA=
3D(')I  DC
 ' 45
In
vitro
3D( ') E< :?s;H;DJ I?P;I 3D(
KBA '  DC 3D( ')I
 DC 7D: 3D( ')I  DC
7D: 3D,(
     7D:  μ& 43D5 /, ,&" 45
Sphaerechinus
granularis
In
vitro
./ H7:?7J?ED !(./ H7:?7J?ED  7D:  A#C !(
  7D:  i&
 J7?B ' ./  A#C
!( i&
45
 I?=D?<?97DJ ?D9H;7I;  I?=D?<?97DJ :;9H;7I; g DE ;<<;9J  7J 7D: 78EL;  J7?B ' . 7H8?JH7HO KD?JI ' :7C7=;: DK9B;? " :7C7=; ?D:;N ! >;7: 7H;7
!" >;7: ?DJ;DI?JO !% >;7: B;D=J> (-& (B?L; J7?B CEC;DJ - J7?B 7H;7 -" J7?B ?DJ;DI?JO -% J7?B B;D=J> -EJ JEJ7B 7H;7 -EJ" JEJ7B ?DJ;DI?JO -EJ% JEJ7B B;D=J>
,&" IF;HC CKJ7=;D?9 ?D:;N vs L?IK7B I9EH?D= &) 7C?DEC;J>OB F>EIF>ED?9 79?: 475) 8;DPE7FOH;D; :B 97:C?KC 9>BEH?:; !( >O:HE=;D F;HEN?:;
UNCORRECTED PROOF
G. Gajski et al. Mutation Research-Reviews in Mutation Research xxx (xxxx) 108398
$H( FEJ7II?KC :?9>HEC7J; &&, C;J>OB C;J>7D;IKB<ED7J; ') D7DEF7HJ?9B;I ) FEI?J?L; 9EDJHEB +(, H;79J?L; ENO=;D IF;9?;I -- JH?8KJOBJ?D 9>BEH?:; ./
KBJH7 L?EB;J 00-) M7IJ;M7J;H JH;7JC;DJ FB7DJ ;<rK;DJ 3D( P?D9 EN?:; 3D,( P?D9 IKBF>7J;
F7HJ?9KB7H B?C?J7J?ED ?J ?I 7FF7H;DJBO 78B; JE I?CKBJ7D;EKIBO ;L7BK7J;
' I?D=B; 7D: :EK8B;IJH7D:;: 8H;7AI ?D >KC7D IF;HC 7D 7FFHE79>
FEJ;DJ?7BBO 7FFB?978B; ?D J>; :EC7?D E< >KC7D <;HJ?B?JO 7D: 7D:HEBE=?
97B F7J>EBE=O 45 &EH; H;9;DJBO EHJ;I KJ?;HH;P ;J 7B 45 7BIE :;
L;BEF;: 7 JME:?C;DI?ED7B JMEJ7?B;: 9EC;J 7II7O M?J> 7 FEII?8?B?JO JE
:?<<;H;DJ?7J; 8;JM;;D I?D=B;IJH7D:;: 7D: :EK8B;IJH7D:;: ' 8H;7AI
?D IF;HC 7D: FEJ;DJ?7B 7FFB?97J?ED ?D <;HJ?B?JO 7II;IIC;DJ
;I?:;I J>; 78EL;C;DJ?ED;: CE:?q97J?EDI I;L;H7B F7F;HI 7BIE :;
I9H?8; J>; KI; E< F= D:E""" 7D:EH >( JH;7JC;DJ JE :;J;9J EN?
:?I;: ' ?D IF;HC E< 8EJ> 7D?C7BI 7D: >KC7DI 45 8KJ
J>;I; 7II7OI IJ?BB D;;: <KHJ>;H EFJ?C?P7J?ED
BJ>EK=> J>; 9EC;J 7II7O ?I KIK7BBO :ED; ED <H;I> I7CFB;I J>?I ?I
?CFH79J?978B; ?D J>; 97I; E< B7H=; ;F?:;C?EBE=?97B >KC7D IJK:?;I 7D: IE
J>; 7FFB?978?B?JO E< KI?D= J>; 9EC;J 7II7O ED <HEP;D IF;HC >7I 8;;D 7I
I;II;: H;IKBJI M?J> 9HOEFH;I;HL;: 7D: <H;I> I7CFB;I 9ECF7H;: M;BB <EH
8EJ> 7D?C7B 7D: >KC7D IF;HC 45
6. The sperm comet assay in animal environmental studies and
reproductive toxicology
->; IF;HC 9EC;J 7II7O <EKD: ?JI HEB; ?D ;DL?HEDC;DJ7B CED?JEH?D=
<HEC ?DL;HJ;8H7J; JE L;HJ;8H7J; IF;9?;I 45 M?J> J>; <EBBEM?D= IJK:?;I
FH;I;DJ;: ?D -78B;I 
6.1. Studies performed on invertebrates
ED9;HD?D= ?DL;HJ;8H7J;I CEBBKI9 IF;HC7JEPE7 >7L; 8;;D H;7:?BO
KI;: <EH 8?ECED?JEH?D= E< 8EJ> <H;I>M7J;H 7D: C7H?D; 7GK7J?9 ;DL?HED
C;DJI I?D9; J>EI; 9;BBI 7H; H;=7H:;: 7I ;N9;BB;DJ 8?EC7HA;HI E< FEBBK
J?ED ,JK:?;I M;H; F;H<EHC;: 8EJ> in vitro 7D: in vivo ED :?<<;H;DJ
IF;9?;I E< 8?L7BL;I IK9> 7I F;FF;HO <KHHEM I>;BB Scrobicularia plana
45 787BED; Haliotis midae 45 7D: EOIJ;HI Crassostrea gigas
45 M?J> J>; L7H?EKI 7?CI E< EFJ?C?I?D= J>; IF;HC 9EC;J 7II7O
FHEJE9EB ?D 8?L7BL;I J;IJ?D= J>; ;<<;9JI E< JEN?97DJI 7D: ;DIKH?D= 7:;
GK7J; <;HJ?B?I7J?ED IK99;II JE C7N?C?P; <7HC FHE:K9J?ED
,JK:?;I >7L; 7BIE 8;;D 97HH?;: EKJ ?D 7DD;B?: IF;HC7JEPE7 D7C;BO
FEBO9>7;J; IF;9?;I E< >?=> ;9EBE=?97B H;B;L7D9; IK9> 7I Arenicola ma-
rina 45 7I M;BB 7I EB?=E9>7;J;I IK9> 7I ;7HJ>MEHCI Eisenia fetida
45 JE J;IJ 7 H7D=; E< 9>;C?97B 7D: F>OI?97B 7=;DJI 8EJ> in vitro
7D: in vivo
HKIJ79;7D IF;HC7JEPE7 97D 7BIE I;HL; 7I 7 =EE: CE:;B ?D ;9EJEN?
9EBE=O 7D: 8?ECED?JEH?D= ,F;HC7JEPE7 E< I;L;H7B 7CF>?FE: 9HKI
J79;7DI IK9> 7I Gammarus fossarum 45 Gammarus elvirae
45 7D: Echinogammarus veneri 45 7I M;BB 7I FH7MDI 7D: I>H?CFI
Macrobrachium rosenbergiiPalaemon serratus 7D: Palaemon longirostris
45 M;H; KI;: ?D EH:;H JE EFJ?C?P; J>; FHEJE9EB EH JE J;IJ :?<<;H
;DJ JEN?97DJI 7I M;BB 7I 78?EJ?9 <79JEHI ?D B78EH7JEHO 9ED:?J?EDI EH ?D J>;
q;B: +;IKBJI 7BIE I>EM;: J>7J IF;HC7JEPE7 7FF;7H;: I?=D?<?97DJBO
CEH; IKI9;FJ?8B; J>7D EE9OJ;I JE =;DEJEN?97DJI "J M7I :;CEDIJH7J;:
J>7J 7 I?=D?<?97DJ 9EHH;B7J?ED ;N?IJI 8;JM;;D J>; B;L;B E< IF;HC '
:7C7=; E< ;NFEI;: 7:KBJI 7D: J>; 78DEHC7B?JO H7J; ?D FHE=;DO ;C
8HOEI &EH;EL;H H;FHE:K9J?ED :;<;9JI M;H; E8I;HL;: 7J B;L;BI E< '
:7C7=; ;N9;;:?D= 7 C?D?C7B J>H;I>EB: M>?9> 9EKB: >7L; I?=D?<?97DJ
9EDI;GK;D9;I <EH J>; FEFKB7J?ED :OD7C?9I E< J>?I ;9EBE=?97BBO H;B;L7DJ
IF;9?;I 45 ,;L;H7B IF;9?;I E< I;7 KH9>?DI Paracentrotus lividus 7D:
Sphaerechinus granularis 7D: J>;?H IF;HC7JEPE7 >7L; 7BIE 8;;D KI;: <EH
J>; 7II;IIC;DJ E< ' :7C7=; H;IKBJ?D= <HEC 8EJ> F>OI?97B 7D: 9>;C?
97B 7=;DJI ?D J>; C7H?D; ;DL?HEDC;DJ 45
->; 9EC;J 7II7O M7I 7BIE ;CFBEO;: ED ?DI;9J IF;HC7JE9OJ;I
D7C;BO ?D <HK?J rO Drosophila JE J;IJ CEB;9KB7H ?DJ;H79J?EDI 8;JM;;D
IOC8?EJ?9 879J;H?7 7D: J>;?H 7D?C7B >EIJI +;IKBJI I>EM;: J>7J :?IHKF
J?ED E< H;:EN >EC;EIJ7I?I B;7:I JE EN?:7J?L; ' :7C7=; ?D IF;HC7JE
9OJ;I E< 0EB879>?7?D<;9J;: Drosophila simulans 45
6.2. Studies performed on vertebrates
I <EH L;HJ;8H7J; IF;9?;I 8;I?:;I ;DL?HEDC;DJ7B IJK:?;I 9ED:K9J;:
?D EH:;H JE 7II;II J>; ?CF79J E< :?<<;H;DJ 9EDJ7C?D7DJI in vitro 7D: in
vivo J>; IF;HC 9EC;J 7II7O >7I 8;;D KI;: M?J> H;=7H: JE 7D?C7B H;FHE
:K9J?ED ;IF;9?7BBO ?D IF;9?;I J>7J >7L; 9ECC;H9?7B L7BK; ->; 9EC;J 7I
I7O M7I 7FFB?;: JE I;7 B7CFH;O Petromyzon marinus IF;HC JE CED?JEH
;<<;9JI E< ;DL?HEDC;DJ7B :?IJKH87D9;I 7D: JE J;IJ J>; ?CF79J E< J>; JEN?
97DJ 8?I7P?H M?J> J>; H;IKBJI E< J>?I IJK:O ?D:?97J?D= 7 =;DEJEN?9 ;<<;9J
->KI J>?I IJK:O :;CEDIJH7J;: J>; IK99;II<KB 7FFB?97J?ED E< J>; 9EC;J
7II7O JE CED?JEH ;DL?HEDC;DJ7B ;<<;9JI ?D I;7 B7CFH;O IF;HC7JEPE7 7D:
FEII?8BO EJ>;H IF;9?;I E< 7D9?;DJ qI> M?J> 79HEIEC7B IF;HC 45 ->;
IF;HC 9EC;J 7II7O >7I 8;;D KI;: JE ;L7BK7J; ;9EJEN?9?JO ?D 7 L7H?;JO E<
<H;I> M7J;H 7D: C7H?D; qI> 7D: 7BIE JE J;IJ J>; FEII?8B; ?CF79J E<
<H;;P?D= 7D: J>7M?D= ED ' ?DJ;=H?JO ;IF;9?7BBO ?D IF;9?;I J>7J >7L;
9ECC;H9?7B L7BK; IK9> 7I JHEKJ Oncorhynchus mykiss, Salmo trutta
45 9>7HH Salvelinus alpinus 45 IJKH=;ED Acipenser
gueldenstaedtiiAcipenser baerii 7D: Acipenser ruthenus 45 I;7
8H;7C Sparus aurata 45 I;7 87II Dicentrarchus labrax 45
r7JqI> Pleuronectes vetulus 7D: Pleuronichthys verticalis 45 O;BBEM
9HE7A;H Pseudosciaena crocea 45 7D: J?B7F?7 Oreochromis niloticus
45
"D 7CF>?8?7DI J>; ;<<;9JI E< IJEH7=; 7D: 9HOEFH;I;HL7J?ED ED IF;HC
' :7C7=; M;H; 7BIE 7II;II;: KI?D= J>; 9EC;J 7II7O "D J>; KHEF;7D
9ECCED <HE= Rana temporaria ?J M7I I>EMD J>7J IF;HC ' :7C7=;
?D9H;7I;: :KH?D= H;<H?=;H7J;: IJEH7=; 7BJ>EK=> J>?I :?: DEJ 7<<;9J IF;H
C7JEPE7B CEJ?B?JO EH <;HJ?B?JO 45 ->; ;<<;9J E< 9HOEFH;I;HL7J?ED ED
IF;HC ' ?DJ;=H?JO ?D J>; 9HE9E:?B; Crocodylus porosus M7I ;N7C
?D;: KI?D= 7 JMEJ7?B;: 9EC;J 7II7O 45 ->; 9EC;J 7II7O M7I 7FFB?;:
JE IF;HC7JEPE7 E< J>; <H?97D 9B7M;: <HE= Xenopus laevis <EH J>; L7B?
:7J?ED E< EJ>;H ' :7C7=?D= 7II7OI 45
->; 9EC;J 7II7O M7I 7BIE KI;: <EH J>; ;L7BK7J?ED E< ' :7C7=; ?D
FEKBJHO 9>?9A;D 7D: JKHA;O IF;HC7JEPE7 :KH?D= 9HOEFH;I;HL7J?ED 7D:
I>EHJJ;HC B?GK?: IJEH7=; M>?9> 7H; 9HK9?7B ?D J;HCI E< 7HJ?q9?7B ?DI;C?
D7J?ED 7I M;BB 7I IJEH7=; ?D 9HOE87DAI 45 <<;9JI E< 9HOEF
H;I;HL7J?ED M;H; 7BIE ;L7BK7J;: ?D LKBJKH;I Gyps fulvus 7D: DE ?D:K9
J?ED E< ' :7C7=; M7I E8I;HL;: :KH?D= J>; FHE9;II M>?9> 9EKB: 8;
8;D;q9?7B ?D J>; =H?<<ED 9EDI;HL7J?ED FHE=H7CI 45
->; IF;HC 9EC;J 7II7O >7I 7BIE 8;;D KI;: ?D F;JI D7C;BO 97JI 7D:
:E=I <EH J>; :;J;9J?ED E< ' :7C7=; ?D J>;?H IF;HC ?D:K9;: 8O 1H7OI
7I M;BB 7I <EH 7II;II?D= I;C;D GK7B?JO 45 ->; IF;HC 9EC;J 7I
I7O M7I 7BIE 9ED:K9J;: ED I;L;H7B EJ>;H :EC;IJ?9 7D?C7BI IK9> 7I
>EHI;I 45 :EDA;OI 45 8KBBI 45 =E7JI
45 I>;;F 45 7D: 8E7HI 45 JE J;IJ I;C;D GK7B?JO
<EH 9HOEFH;I;HL7J?ED 7D: 7HJ?q9?7B ?DI;C?D7J?ED
->; IF;HC 9EC;J 7II7O >7I 8;;D 7FFB?;: JE M?B: 7D?C7BI 7I M;BB
,F;HC ' ?DJ;=H?JO M7I ;L7BK7J;: ?D I;L;H7B C;J7J>;H?7D IF;9?;I
D7C;BO ?D ;9>?:D7 Tachyglossus aculeatus MEC87J Vombatus ursinus
AE7B7 Phascolarctos cinereus 7D: A7D=7HEE Macropus giganteus ?D:?
97J?D= J>7J J>; IF;HC ' E< J>; C7HIKF?7B IF;9?;I ?I CEH; I;DI?J?L; JE
EN?:7J?L; IJH;II ?DIKBJI 9ECF7H;: JE ;KJ>;H?7D IF;9?;I 45
I <EH B7H=; M?B:B?<; C7CC7BI J>; IF;HC 9EC;J 7II7O M7I ;CFBEO;:
JE 7II;II ' :7C7=; ?D J>H;; H>?DE9;HEI IF;9?;I Diceros bicornisRhi-
noceros unicornis 7D: Ceratotherium simum 7D: :?I9EL;H;: J>; FH;I;D9;
E< ' :7C7=; I>EHJBO 7<J;H J>7M?D= J>; IF;HC 45  I?C?B7H ;<<;9J
M7I <EKD: <EH CEDA;O Macaca mulatta IF;HC <EBBEM?D= <H;;P
?D=J>7M?D= 45 D7BOI?I E< IF;HC 9>HEC7J?D IJHK9JKH; ?D <EN;I
Alopex lagopus 7D: Vulpes vulpes I>EM;: J>7J J>; =;D;J?9 C7J;H?7B E<
UNCORRECTED PROOF
G. Gajski et al. Mutation Research-Reviews in Mutation Research xxx (xxxx) 108398
Table 2
->; IF;HC 9EC;J 7II7O <EH J>; ;L7BK7J?ED E< ' :7C7=; ?D L;HJ;8H7J;I
D?C7B -OF;
E<
IJK:O
=;DJIJH;IIEH ED9;DJH7J?ED H7D=; )7H7C;J;HI
J;IJ;:
+;IFEDI; +;<;H;D9;I
Cyclostomes Petromyzon
marinus
In
vitro,
in
vivo
!( ./ 8?I7P?H IJEH7=;
9ED:?J?EDI
!(  7D:  μ& ./  7D:
 #C IJEH7=; 9ED:?J?EDI  7D: 
:7OI 8?I7P?H  C=C%
 >;7:
' /, ,
(-&
!( i&
./  #C IJEH7=;
9ED:?J?EDI  :7OI 8?I7P?H
45
Fishes Oncorhynchus
mykiss
In
vitro
HOEFH;I;HL7J?ED H;;P?D=J>7M?D=  J7?B '
(-& 45
In
vitro
HOEFH;I;HL7J?ED H;;P?D=J>7M?D= -&  E<
:7C7=;:
DK9B;?
45
In
vitro
./ !(./ ?HH7:?7J?ED  C09C !( 
7D:  C&  i& !(7I )
 J7?B '
(-& ./ !( 45
In
vitro
!= : !=   7D:  C=% :  
7D:  C=%
 >;7: ' !=  C=% :
 C=%
45
In
vitro
HOEFH;I;HL7J?ED !(7I
)
H;;P?D=J>7M?D= !( C&  J7?B ' 45
In
vitro
HOEFH;I;HL7J?ED KI?D=
;== OEBA 7D: %% 7I
7::?J?L;I !(7I )
H;;P?D=J>7M?D= !( C&  J7?B ' 45
In
vitro
HOEFH;I;HL7J?ED KI?D=
;== OEBA 7D: %% 7I
7::?J?L;I !(7I )
H;;P?D=J>7M?D= !( C&  J7?B ' 45
Salmo trutta In
vitro
HOEFH;I;HL7J?ED !(7I
)
H;;P?D=J>7M?D=  J7?B '  g :;F;D:;DJ ED J>; BE97J?ED 45
In
vivo
&&, &&,  C=A= 8E:O M;?=>J FH;L?EKIBO
:?IIEBL;: ?D M7HC;:KF 9E9EDKJ E?B  C%
E?BA= 8E:O M;?=>J
 J7?B ' 45
Salvelinus
alpinus
In
vivo
&&, &&,  C=A= 8E:O M;?=>J FH;L?EKIBO
:?IIEBL;: ?D M7HC;:KF 9E9EDKJ E?B  C%
E?BA= 8E:O M;?=>J
 J7?B ' 45
Acipenser
gueldenstaedtii
In
vitro
,>EHJJ;HC B?GK?:
IJEH7=;
;HE8?9 9ED:?J?EDI 7J  d  J7?B '
(-& :;F;D:;DJ ED J>; J?C; E< IJEH7=; 45
Acipenser
baerii
In
vitro
,>EHJJ;HC B?GK?:
IJEH7=;
;HE8?9 9ED:?J?EDI 7J  d  J7?B '
(-& :;F;D:;DJ ED J>; J?C; E< IJEH7=; 45
Acipenser
ruthenus
In
vitro
KHEGK?DED;    7D:  i&  J7?B '
(-&  i& 45
Sparus aurata In
vitro
HOEFH;I;HL7J?ED H;;P?D=J>7M?D=  J7?B '
(-& 45
Dicentrarchus
labrax
In
vitro
HOEFH;I;HL7J?ED
HOEFHEJ;9J7DJI
H;;P?D=J>7M?D=  J7KH?D; 7D:
>OFEJ7KH?D;
 J7?B ' 9HOEFH;I;HL7J?ED
9HOEFHEJ;9J7DJI
45
In
vitro
HOEFH;I;HL7J?ED H;;P?D=J>7M?D=  J7?B '
-& 45
Pleuronectes
vetulus
In
situ
07IJ;M7J;H EKJ<7BB (H7D=; EKDJO  ., -& 45
Pleuronichthys
verticalis
In
situ
07IJ;M7J;H EKJ<7BB (H7D=; EKDJO  ., -& 45
Pseudosciaena
crocea
In
vitro
HOEFH;I;HL7J?ED
9HOEFHEJ;9J7DJ
H;;P?D=J>7M?D= &,(     
7D:  
% -% +
9E; ↑ ≥   45
Oreochromis
niloticus
In
vivo
EHED     7D:  C=% /, 7J  C=% 45
Oreochromis
niloticus
In
vivo
K,(  7D:  C=A=  J7?B ' 45
Oreochromis
niloticus
In
vitro
HOEFH;I;HL7J?ED
9HOEFHEJ;9J7DJ =BO9;HEB
C;J>7DEB 7D: &,(
H;;P?D=J>7M?D= /, &,( :;F;D:;DJ ED J>;
9HOEFHEJ;9J7DJ
45
Amphibians Rana
temporaria
In
vitro
HOEFH;I;HL7J?ED ,JEH7=; 9ED:?J?EDI /, :;F;D:;DJ ED J>; :7OI E< IJEH7=; 45
Xenopus laevis In
vitro
/7B?:7J?ED E< J>; IF;HC
9>HEC7J?D :?IF;HI?ED J;IJ
?D H;B7J?ED JE 9EC;J 7II7O
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Reptiles Crocodylus
porosus
In
vitro
HOEFH;I;HL7J?ED H;;P?D=J>7M?D= /, 45
Birds Gallus gallus
domesticus
In
vitro
HOEFH;I;HL7J?ED
9HOEFHEJ;9J7DJI
H;I> 7D: <HEP;D IF;HC7JEPE7
9HOEFHEJ;9J7DJI =BO9;HEB   =BO9;HEB 
 7D: JH;>7BEI; JH>  CCEB% & 
&  7D: JH>  CCEB%
 J7?B '
-% (-& % & C;:?KC 45
continued on next page
UNCORRECTED PROOF
G. Gajski et al. Mutation Research-Reviews in Mutation Research xxx (xxxx) 108398
-78B;  continued
D?C7B -OF;
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gallopavo
In
vitro
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Mericanel
della Brianza
In
vitro
HOEFH;I;HL7J?ED H;;P?D=J>7M?D=  d  d  &
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In
vitro
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Gyps fulvus In
vitro
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Mammals Felis silvestris
catus
In
vitro
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viverrinus
In
vitro
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Canis lupus
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In
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In
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Equus asinus In
vitro
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In
vitro
%E97B?P7J?ED E< 7BA7B?
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Bos taurus In
vitro
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In
vitro
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In
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45
In
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In
vitro
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&,( JE H;FB79; =BO9;HEB
45
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In
vitro
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H;;P?D=J>7M?D= B;9?J>?D      7D:
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45
Capra hircus
ancryrensis
In
vitro
HOEFH;I;HL7J?ED
H7s?DEI; C;J>?ED?D;
H;;P?D=J>7M?D= H7s?DEI;   7D:
 C& C;J>?ED?D;   7D:  C&
/, . :;F;D:;DJ ED J>;
9ED9;DJH7J?ED
45
Ovis aries In
vitro
HOEFH;I;HL7J?ED
H7s?DEI; >OFEJ7KH?D; 
C?NJKH;
H;;P?D=J>7M?D= H7s?DEI;  C&
>OFEJ7KH?D;  C& +! C?NJKH;
   C&
/, 45
In
vitro
HOEFH;I;HL7J?ED "DrK;D9; E< IF;HC 9ED9;DJH7J?ED /, . :;F;D:;DJ ED J>;
9ED9;DJH7J?ED
45
continued on next page
UNCORRECTED PROOF
G. Gajski et al. Mutation Research-Reviews in Mutation Research xxx (xxxx) 108398
-78B;  continued
D?C7B -OF;
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In
vitro
HOEFHEJ;9J7DJI =BO9;HEB
  7D: ;J>OB;D; =BO9EB
  JH;>7BEI; -
   C& - 
 C& -      C& -
7D:   C& -
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9HOEFHEJ;9J7DJI ?D 9EC8?D7J?ED M?J>
-
45
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vitro
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JH;>7BEI; OEBA
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OEBA   LL
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In
vitro
HOEFH;I;HL7J?ED %%
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In
vitro
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aculeatus
In
vitro
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Vombatus
ursinus
In
vitro
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IF;9?;I ?I CEH; I;DI?J?L; JE EN?:7J?L;
IJH;II J>7D J>; IF;HC7JEPE7 E<
;KJ>;H?7D IF;9?;I
45
Macropus
giganteus
In
vitro
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IF;9?;I ?I CEH; I;DI?J?L; JE EN?:7J?L;
IJH;II J>7D J>; IF;HC7JEPE7 E<
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45
Phascolarctos
cinereus
In
vitro
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IF;9?;I ?I CEH; I;DI?J?L; JE EN?:7J?L;
IJH;II J>7D J>; IF;HC7JEPE7 E<
;KJ>;H?7D IF;9?;I
45
In
vitro
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Diceros
bicornis
In
vitro
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Rhinoceros
unicornis
In
vitro
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simum
In
vitro
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Macaca
mulatta
In
vitro
HOEFH;I;HL7J?ED H;;P?D=J>7M?D=  J7?B ' 45
Alopex
lagopus
In
vitro
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45
Vulpes vulpes In
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HOEFH;I;HL7J?ED 7J  d /,  >;7:
' :;F;D:;DJ ED J>; J?C; 7<J;H
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45
Tursiops
truncatus
In
vitro
/7B?:7J?ED :?s;H;DJ ?D9K87J?ED J?C;I /, ↑↓ :;F;D:;DJ ED J>; IF;HC
CEHF>EJOF;
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J>; IF;HC E< I?BL;H <EN;I M7I CEH; IJ78B; 9ECF7H;: JE J>; 7H9J?9 ED;I
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M7I KI;: <EH J>; ;L7BK7J?ED E< IF;HC ' ?DJ;=H?JO E< :EBF>?DI Tur-
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F>OI?97B 7=;DJI "D C?9; J>; 7II7O M7I F;H<EHC;: ?D EH:;H JE J;IJ J>; ;<
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45 D7DEJK8;I 45 9?=7H;JJ; ICEA; 45 1H7OI 45
=7CC7 7D: H7:?E<H;GK;D9O H7:?7J?ED 45 ->; IF;HC 9EC;J 7II7O
M7I KI;: JE J;IJ FHEJ;9J?L; ;<<;9JI E< :?<<;H;DJ IK8IJ7D9;I 7D: D7JKH7B
FHE:K9JI 45 7I M;BB 7I J>; ?DrK;D9; E< 7=?D= 45 ,?C?B7H
IJK:?;I M;H; 9ED:K9J;: ?D H7J IF;HC 7D: ;L7BK7J;: J>; ' :7C7=?D=
;<<;9JI E< 7HI;D?9 45 C;H9KHO 45 9?=7H;JJ; ICEA; 7D: D?9EJ?D;
45 7DJ?:;FH;II7DJI 45 7DJ?;F?B;FJ?9 :HK=I 45 7DJ?
97D9;H :HK=I 45 7D78EB?9 IJ;HE?:I 45 F;IJ?9?:;I 45 8?IF>;
DEB  45 FEBO9>BEH?D7J;: 8?F>;DOBI 45 D7DEF7HJ?9B;I 45
:;FB;J;: KH7D?KC 45 ;B;9JHEC7=D;J?9 ?HH7:?7J?ED 45 ->; IF;HC
9EC;J 7II7O M7I KI;: JE J;IJ FHEJ;9J?L; ;<<;9JI E< :?<<;H;DJ IK8IJ7D9;I
7D: D7JKH7B FHE:K9JI 7BIE ?D H7JI 45 7I M;BB 7I
J>; ?CF79J E< :?;J 7D: E8;I?JO 45 ;I?:;I C?9; 7D: H7JI J>;H;
7H; 7BIE I;L;H7B H;FEHJI E< IJK:?;I F;H<EHC;: ED >7CIJ;HI 7D: H788?JI
45
I :;I9H?8;: 78EL; J>; 9EC;J 7II7O ?I ;NJ;DI?L;BO KI;: ?D 7D?C7BI
;IF;9?7BBO M?J> H;=7H: JE ;L7BK7J?D= J>; ?CF79J E< 9HOEFH;I;HL7J?ED ED
IF;HC ' ?DJ;=H?JO 8;97KI; J>; FHE9;II E< 9HOEFH;I;HL7J?ED ?I 9HK9?7B
<EH 7HJ?q9?7B ?DI;C?D7J?ED 7D: <EH J>; 9EDI;HL7J?ED E< =;D;J?9 H;IEKH9;I
?D 9HOE87DAI ,F;HC ' ?DJ;=H?JO C7O 8; ?CF7?H;: :KH?D= 9HOEFH;I;H
L7J?ED >EM;L;H J>; :7C7=;I ?D:K9;: ED DK9B;7H ' 7H; C?DEH 9EC
F7H;: JE J>; :7C7=;I FHE:K9;: ED EJ>;H 9;BBKB7H 9>7H79J;H?IJ?9I IK9> 7I
C;C8H7D; ?DJ;=H?JO 7D: CEJ?B?JO BJ>EK=> J>;H; 7H; H;IKBJI ?D:?97J?D=
J>7J 7D ?D9H;7I; ?D ' :7C7=; E99KHI :KH?D= 9HOEFH;I;HL7J?ED :;
F;D:?D= ED J>; 9ED:?J?EDI 7I M;BB 7I IF;9?;I KI;: J>;O 7BIE IK==;IJ 7
BEM I;DI?J?L?JO E< L;HJ;8H7J; IF;HC7JEPE7 JE ' 8H;7A7=; J>7J I>EKB:
DEJ 8; 9EDI?:;H;: 7I 7 C7@EH 97KI; E< IF;HC ?D@KH?;I :KH?D= 9HOEFH;I;H
L7J?ED ';L;HJ>;B;II =?L;D J>; KDKIK7BBO >?=> ?D9?:;D9; E< ' :7C
UNCORRECTED PROOF
G. Gajski et al. Mutation Research-Reviews in Mutation Research xxx (xxxx) 108398
Table 3
->; IF;HC 9EC;J 7II7O <EH J>; ;L7BK7J?ED E< ' :7C7=; ?D B78EH7JEHO HE:;DJI
D?C7B -OF;
E<
IJK:O
=;DJIJH;IIEH ED9;DJH7J?ED H7D=; )7H7C;J;HI J;IJ;: +;IFEDI; +;<;H;D9;I
"+
C?9;
In vivo %;7: ;NFEIKH;    7D:  B;7: 79;J7J;  J7?B ' -% J7?B
CEJ?ED (-& ↑ ≥   45
In vivo &;J>7C?:EF>EI J;9>D?97B &-J 7D: 9ECC;H9?7B &-9 
 7D:  C=A= 8M:7O :7OI
 J7?B ' -& ↑ ≥  C=A= 8M:7O :7OI 45
In vivo &;J>7C?:EF>EI  7D:  C=A=8M?F:7O :7OI  J7?B ' -& 45
In vivo &0'- DEDOBF>;DEB ')
!(7I )
&0'-  C=A= 8M ')  C=A= 8M 
9EC8?D;: !( μCEB%
 J7?B ' (-& g ')&0'- 9EC8?D;: 45
In vivo >HED?9 79HOB7C?:;
;NFEIKH; 7C;B?EH7J?ED 8O
H;IL;H7JHEB
9HOB7C?:;  C=A= 8E:OM;?=>J:7O
H;IL;H7JHEB  C=A= 8MM;;A
 J7?B ' 9EC8?D;: 45
,M?II C?9; In vivo >HED?9 79HOB7C?:;
;NFEIKH;
    7D:  i=C% ;GK?L7B;DJ JE
 C=A= 8M:7O
 J7?B ' -& ↑ ≥  i=C% 45
In vivo &;J>EJH;N7J;    7D:  C=A= -%  J7?B ' -&
(-& 45
In vivo HJ;IKD7J;  7D:  C=A= 8M  J7?B ' (-& /, " 45
In vivo +7:?E<H;GK;D9O H7:?7J?ED
C;B7JED?D  9EC8?D;:
 &!P &%  C=A= 8M:7O  >;7: '  J7?B
' "  ++ g 9EC8?D;: 45
)P>,<?I
C?9;
In vivo ) 1H7OI )   7D:  C=A= 1H7OI  O
9EC8?D7J?ED  O C=A= 8M )
 J7?B ' 45
 C?9; In vivo ) 7=?D=  C=A=  J7?B ' -& g 45
# C?9; In vivo ?=7H;JJ; ICEA; ;9B7H;: 9EDJ;DJ E<  C= J7H 7D:  C=
D?9EJ?D;
 J7?B ' 45
% #
C?9;
In vivo 1H7OI O J7?B ' 45
0?IJ7H H7JI In vivo ?=7H;JJ; ICEA;
7C;B?EH7J?ED 8O 3DB
9?=7H;JJ; ICEA;  9?=7H;JJ;I F;H :7O 3DB
 C=A=:7O  9EC8?D;:
 J7?B ' -& 9?=7H;JJ; ICEA; g 3DB
9EC8?D;:
45
In vivo '?9EJ?D; ;s;9J ED
E<<IFH?D=
)H;=D7DJ 7D: B79J7J?D= H7JI M;H; D?9EJ?D;
;NFEI;:  C=A=:7O
 J7?B ' -% -& (-& 45
In vivo ,;HJH7B?D;   7D:  C=A=:7O -& 45
In vivo ?J7BEFH7C >O:HE8HEC?:;   7D:  C=A=:7O -& 45
In vivo %;L;J?H79;J7C   7D:  C=A=:7O -& ↑ ≥  C=A=:7O 45
In vivo '7D:HEBED; :;97DE7J;
7C;B?EH7J?ED 8O J7KH?D;
'  C=A=M;;A -  C=A=:7O 
9EC8?D;:
 J7?B ' -% -&
(-& ' g - 9EC8?D;: 45
In vivo ?FHED?B   7D:  C=A=:7O  J7?B ' -% -&
(-& . ↑ ≥  C=A=:7O 45
In vivo =')I  EH  C=A= E<  DC EH  DC =')I  J7?B '  >;7:
' -%  g I?P; :EI; 7D: J?C;
:;F;D:;DJ
45
In vivo ;FB;J;: .H7D?KC  7D:
 =;D;H7J?ED
 7D:  C=A=:7O  J7?B ' -% -&
(-& 45
In vivo B;9JHEC7=D;J?9 q;B: & CE8?B; F>ED; H7:?7J?ED  &!P  J7?B ' -% -&
(-&  B7H=; DKC8;H E<
EJ>;H F7H7C;J;HI
45
,FH7=K;
7MB;O
H7JI
In vivo ENEHK8?9?D   7D:  C=A= M;;ABO  J7?B ' -% -&
J7?B;: 9;BBI ↑ ≥  C=A=M;;ABO 45
In vivo HI;D?9 7C;B?EH7J?ED 8O
GK;H9;J?D
I  FFC *.  C=A= 8M %  >;7: ' -% 
J7?B ' -& (-& I g 9EC8?D;: 45
In vivo !=B 7C;B?EH7J?ED 8O
Chenopodium album Linn.
7D: /?J 
!=B C=A= 8M /?J   C=A= 8M
 7B8KC  C=A= 8M
!%  >;7: ' -% 
J7?B ' -& !=B g C. album g
9EC8?D;: :;F;D:;DJ ED J>;
:;I9H?FJEH
45
In vivo ) 7C;B?EH7J?ED 8O &% )  C=A= 8M :7O &%  C=A=
8M:7O
 J7?B ' -% ) g &% 9EC8?D;: 45
In vivo HE9BEH  ?CF79J E< ,; HE9BEH  C=A= ,;  C=A= 7D:
 C=A= :?;J
 J7?B ' HE9BEH ,;   ,; g
,;,   ,;,
45
In vivo !?=><7J :?;J FHEJ;9J?L;
;<<;9J E< FHE8?EJ?9I
'EHC7B IJ7D:7H: :?;J  <7J MM >?=><7J
:?;J   <7J MM >?=><7J :?;J  
FHE8?EJ?9I MM
 J7?B ' -% -&
(-& >?=><7J :?;J FHE8?EJ?9I 45
In vivo ,-3?D:K9;: :?78;J?9 H7JI
7C;B?EH7J?ED 8O J;BC?I7HJ7D
-
,-3  C=A= -   7D:  C=A=:7O  J7?B ' -% -&
(-& ,-3 g - ,-3  - 45
!EBJPC7D
H7JI
In vivo )  μ= 7D:  C=A=8M  J7?B ' -% -&
(-& ↑ ≥  C=A=8M 45
3K9A;H H7J In vivo (8;I?JO %;7D vs <7J  J7?B ' -% -&
(-& <7J 45
Mesocricetus
auratus
In vivo )HEJ;9J?ED E< IF;HC ' 8O
799;IIEHO I;N =B7D:
I;9H;J?EDI
')!?D:K9;: (,     7D:
 CCEB%
-& &7B; 799;IIEHO I;N =B7D:
I;9H;J?EDI 97D FH;I;HL; J>;
?DJ;=H?JO E< J>; IF;HC =;DEC;
45
In vivo )HEJ;9J?L; HEB; E< ,( -
)N EH ,!)N
')!?D:K9;: (,     7D:
 CCEB%
DKC8;H E< 9EC;JI F;H 
 IF;HC :;F;D:;DJ ED J>; ;DPOC; 45
continued on next page
UNCORRECTED PROOF
G. Gajski et al. Mutation Research-Reviews in Mutation Research xxx (xxxx) 108398
-78B;  continued
D?C7B -OF;
E<
IJK:O
=;DJIJH;IIEH ED9;DJH7J?ED H7D=; )7H7C;J;HI J;IJ;: +;IFEDI; +;<;H;D9;I
Oryctolagus
cuniculus
In
vivo/in
vitro
+",. e +;L;HI?8B;
"D>?8?J?ED E< ,F;HC .D:;H
K?:7D9; !(7I )
/7I E99BKI?ED M?J> +",. e <EH 7D: 
CEDJ>I H;L;HI7B M?J> &,( 7D: '7!(7<J;H
 7D:  CEDJ>I !( μ&
 J7?B ' % (-& g !( 45
 I?=D?<?97DJ ?D9H;7I;  I?=D?<?97DJ :;9H;7I; g DE ;<<;9J  7J 7D: 78EL;  >;7: '  J7?B ' . 7H8?JH7HO KD?JI % 9EC;J B;D=J>  :7C7=; <H;GK;D9O
" :7C7=; ?D:;N !% >;7: B;D=J> (-& (B?L; J7?B CEC;DJ -& J7?B ;NJ;DJ CEC;DJ -% J7?B B;D=J> -& J7?B CEC;DJ vs L?IK7B I9EH?D= ) 8?IF>;DEB  -
97J7B7I; ) 9O9BEF>EIF>7C?:; &,( :?C;J>OB IKB<EN?:; & ;B;9JHEC7=D;J?9 q;B: )N =BKJ7J>?ED; F;HEN?:7I; ,! =BKJ7J>?ED; !( >O:HE=;D F;HEN?:;
!=B C;H9KHO 9>BEH?:; &% C;B7JED?D &0'- CKBJ?M7BB;: 97H8ED D7DEJK8;I '7!( IE:?KC 8?97H8ED7J; ') D7DEF7HJ?9B;I (, EN?:7J?L; IJH;II ) FEI?
J?L; 9EDJHEB ,( IKF;HEN?:; :?ICKJ7I; ,-3 IJH;FJEPEJE9?D
7=; ?D J>; IF;HC E< IEC; ?D:?L?:K7B 7D?C7BI 7<J;H 9HOEFH;I;HL7J?ED 7D:
J>7M?D= J>; IJ7D:7H:?P7J?ED E< 7 C;J>E:EBE=O JE 7II;II IF;HC '
:7C7=; ?D IK9> 7D?C7BI 9EKB: 9EDJH?8KJ; JE C7B; H;FHE:K9J?L; C7D7=;
C;DJ E< >?=>BO ;D:7D=;H;: IF;9?;I 45
7. The sperm comet assay in human biomonitoring and
reproductive toxicology
,JK:?;I H;FEHJ?D= H;IKBJI <HEC IF;HC 9EC;J 7II7O KI;: ;?J>;H <EH J>;
FKHFEI; E< >KC7D 8?ECED?JEH?D= EH <EH >KC7D H;FHE:K9J?L; JEN?9EB
E=O 7H; FH;I;DJ;: ?D -78B;I  7D: 
7.1. Use of the sperm comet assay in human biomonitoring
EH J>; FKHFEI; E< >KC7D 8?ECED?JEH?D= J>; IF;HC 9EC;J 7II7O >7I
8;;D ;CFBEO;: JE J;IJ J>; ;<<;9JI E< ;?J>;H ;DL?HEDC;DJ7B EH E99KF7
J?ED7B ;NFEIKH; JE >;7LO C;J7BI 45 8EHED 45 8;DP;D;
45 IJOH;D; 45 79HOBED?JH?B; 45 F>J>7B7J;I 45
F;IJ?9?:;I 45 FEBO9O9B?9 7HEC7J?9 >O:HE97H8EDI 45 ?ED?I?D=
H7:?7J?ED 45 I>E9AM7L; B?J>EJH?FIO 45 7I M;BB 7I IEC; B?<;IJOB;
>78?JI IK9> 7I ICEA?D= 45 IB;;F :KH7J?ED 45 EH C?9HEDKJH?
;DJI ?DJ7A; 45 ,9>C?: ;J 7B 45 ?DL;IJ?=7J;: J>; 7IIE9?7J?EDI 8;
JM;;D C7B; 7=; 7D: IF;HC ' :7C7=; 7D: J>; ?DrK;D9; E< I;L;H7B
B?<;IJOB; <79JEHI ?D >;7BJ>O C7B;I 7D: <EKD: J>7J EB:;H C;D >7L; ?D
9H;7I;: IF;HC ' :7C7=; "D 7::?J?ED C;D M?J> IK8IJ7DJ?7B :7?BO
97<<;?D; 9EDIKCFJ?ED >7: ?D9H;7I;: IF;HC ' :7C7=; ->; 7KJ>EHI
9ED9BK:;: J>7J ?D:K9;: ' :7C7=; 9EKB: 8; 9EDL;HJ;: JE 9>HECEIE
C7B 78;HH7J?EDI 7D: =;D; CKJ7J?EDI 7<J;H <;HJ?B?I7J?ED >;D9; ?D9H;7I?D=
J>; H?IAI E< :;L;BEFC;DJ7B :;<;9JI 7D: =;D;J?9 :?I;7I;I 7CED= E<<
IFH?D= ->; 78EL; IJK:?;I 7D: EJ>;H 7L7?B78B; B?J;H7JKH; ?D:?97J; J>7J
I;L;H7B ;DL?HEDC;DJ7B 7D: E99KF7J?ED7B 7=;DJI 7I M;BB 7I 9;HJ7?D
B?<;IJOB; >78?JI IK9> 7I ICEA?D= C7O 7<<;9J IF;HC ' ?DJ;=H?JO
45
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BEM?D= 97D9;H :?7=DEI?I H;L;7B?D= ?D9H;7I;: ' :7C7=; ?D 97D9;H F7
J?;DJI 9ECF7H;: M?J> <;HJ?B; >;7BJ>O :EDEHI 45 7D: 7 9ECF7H7J?L;
IJK:O E< <;HJ?B; 7D: ?D<;HJ?B; C;D ?D:?97J;: >?=>;H IF;HC ' 8H;7A7=;
?D J>; ?D<;HJ?B; IK8@;9JI 45 ?<<;H;DJ 9B?D?97B =HEKFI E< F7J?;DJI
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<;HJ?B; :EDEHI 45 "D 7::?J?ED J>; 9EC;J 7II7O M7I KI;: JE 7I
I;II J>; :;=H;; E< ' :7C7=; ?D IF;HC 97J;=EH?P;: 8O &EJ?B; ,F;HC
(H=7D;BB; &EHF>EBE=O N7C?D7J?ED &,(& =H7:;I I>EM?D= J>7J
BEM;H &,(& =H7:?D= 9EHH;B7J;: M?J> BEM;H IF;HC ' :7C7=;
->;H;<EH; J>; E8I;HL7J?ED E< IF;HC DK9B;7H L79KEB;I KI?D= H;7BJ?C;
EFJ?97B C?9HEI9EFO M?J>EKJ FH;9?I; ' :7C7=; ;N7C?D7J?ED ?I DEJ
IK<q9?;DJ <EH EFJ?C7B IF;HC I;B;9J?ED <EH "," 45
7.2. Use of the sperm comet assay for in vitro studies and cryopreservation
research
"D EH:;H JE ?DL;IJ?=7J; J>; JEN?9EBE=?97B FHEF;HJ?;I E< :?<<;H;DJ
9>;C?97B 7D: F>OI?97B 7=;DJI 7D: J>; 7IIE9?7J?ED 8;JM;;D IK9> ;NFE
IKH;I 7D: C7B; H;FHE:K9J?L; JEN?9?JO IF;HC >7L; 8;;D ;NFEI;: in vitro
+;=7H:?D= H;FHE:K9J?L; JEN?9?JO IJK:?;I >7L; <E9KI;: ED ;<<;9JI E<
8;DPE4a5FOH;D; 45 &&, 45 F;HrKEHE7BAOB 7D: FEBOrKEHE7BAOB
IK8IJ7D9;I 45 ;IJHE=;D?9 9ECFEKD:I 45 7D7;IJ>;J?9I 45 7DJ?
97D9;H :HK=I 45 7DJ?C7B7H?7B :HK=I 45 <EE: 7::?J?L;I 45
F;IJ?9?:;I 45 D?9EJ?D; 45 :H?DA?D= M7J;H :?I?D<;9J?ED 8O
FHE:K9JI 45 D7DEF7HJ?9B;I 45 =7CC7 H7OI 45 ?Dr7C
C7JEHO <79JEHI 45 7D: EJ>;H :?;J7HO;DL?HEDC;DJ7B 9ECFEKD:I
45 I;; -78B; 
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?:7J?L; IJH;II ?I J>; C7@EH ;<<;9JEH ?D ' :7C7=; :KH?D= I7CFB; 9HOEF
H;I;HL7J?ED 45 ->KI J>;H; ?I 7 9;HJ7?D H?IA E< :;9H;7I;: <;HJ?B
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9HOEFH;I;HL7J?L; =7L; CEIJ I?C?B7H H;IKBJI JE <H;I> >KC7D I;C;D I7C
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FHEJ;9J?L; ;<<;9JI E< :?<<;H;DJ IK8IJ7D9;I 7=7?DIJ 9HOEFH;I;HL7J?ED
9>;C?97BI 7D: +(, ?D:K9;: ' :7C7=; ?D IF;HC ,;L;H7B 9ECFEKD:I
I>EM;: 7 =EE: 97F78?B?JO JE FHEJ;9J J>; =;D;J?9 IJ78?B?JO E< >KC7D IF;H
C7JEPE7 IK9> 7I 79;JOB…97HD?J?D; 45 =7D=B?EI?:;I 45
=;D?IJ;?D 45 EB;EOB;J>7DEB7C?:; 45 7D: r7LEDE?:I 45
7I M;BB 7I L?J7C?DI  7D:  45
8. Biological and clinical significance of DNA damage in sperm
' :7C7=; 7D: ?JI 799KCKB7J?ED 9EDIJ?JKJ; 7 9ECCED FHEF;HJO E<
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EH ?DIJ7D9; :7C7=; ?D IF;HC :K; JE 7 I?D=B; ;NFEIKH; M?BB :?I7FF;7H
M?J>EKJ 7DO ;<<;9JI ?< J>7J =;D;H7J?EDE< IF;HC ?I DEJ KI;: <EH C7A?D=
FHE=;DO 7C7=; ?D IF;HC7JE=ED?7 IF;HCIJ;C 9;BBI 97D >EM;L;H
B;7: JE CKJ7J?EDI J>7J 97D ?D J>;EHO 8; JH7DIC?JJ;: JE 7DO E<<IFH?D= 7<
J;H J>; CKJ7J?ED E99KHH;: 8;97KI; IF;HC :;H?L;: <HEC IK9> IF;HC7JE
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=ED?7 7H; ' H;F7?H FHEq9?;DJ 7D: J>; ?D?J?7B ' :7C7=; ?D IF;H
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IF;HC BEI; J>; 78?B?JO JE H;F7?H J>;?H ' :KH?D= C7JKH7J?ED IEC;
JOF;I E< B;I?EDI ?D IF;HC ' C7O 8; H;F7?H;: ?D J>; EE9OJ; "< J>?I ?I
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B;7:?D= JE CKJ7J?EDI E< J>; C7B; =;DEC; ?D 7BB :;I9;D:;DJ 9;BBI I 7 H;
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:7C7=; :;H?L;: <HEC J>; C7B; =;DEC; C7O B;7: JE 7D ;7HBO 78EHJ?ED
KHJ>;HCEH; IF;HC ' :7C7=; 7D: <H7=C;DJ7J?ED C7O ?D =;D;H7B 8;
J>; 97KI; E< DKC;HEKI :?I;7I;I ?D9BK:?D= 9>?B:>EE: 97D9;H
45

UNCORRECTED PROOF
G. Gajski et al. Mutation Research-Reviews in Mutation Research xxx (xxxx) 108398
Table 4
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=;DJIJH;IIEH NFEIKH;I;JJ?D= )7H7C;J;HI
J;IJ;:
+;IFEDI; +;<;H;D9;I
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-% -&
!=  '?  &D
45
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E99KF7J?ED7B 8EHED
;NFEIKH;
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EHED;NFEI;: MEHA;HI !(
7I )
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(99KF7J?ED7B 8;DP;D;
;NFEIKH;
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(99KF7J?ED7B IJOH;D;
;NFEIKH;
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(-& 45
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;NFEIKH;
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(99KF7J?ED7B 79HOBED?JH?B;
' ;NFEIKH;
NFEIKH;J?C; M7I  O ->; C;7D 9ED9;DJH7J?ED E< ' M7I  h  C=C7J EF;H7J?ED I?J;I -% H7J;I E<
9EC;J IF;HC 45
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F>J>7B7J;I
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E9JOB F>J>7B7J; &() CEDEC;J>OB:?C;J>OB>;NOB ?IEDEDOB F>J>7B7J; &"') 7D:
CEDE9O9BE>;NOB F>J>7B7J; &!)
 J7?B '
-% -& 45
,;C;D F>J>7B7J; C;J78EB?J;I  F>J>7B7J; C;J78EB?J;I 4CEDEC;J>OB F>J>7B7J; &&) CEDE;J>OB F>J>7B7J; &) &) CEDED
E9JOB F>J>7B7J; &() CEDE8;DPOB F>J>7B7J; &P) &!) CEDE;J>OB>O:HENO>;NOB
F>J>7B7J; &!!) 7D: CEDE;J>OBENE>;NOB F>J>7B7J; &(!)5
 J7?B '
-& 45
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F>J>7B7J;I
!7?H ) 7D: KH?D7HO F>J>7B7J;  J7?B '
(-&
g 45
(99KF7J?ED7B <;DL7B;H7J;
;NFEIKH;
)HE:K9J?ED 7H;7 MEHA;HI  J7?B '
-&  45
.H?D7HO B;L;BI E< ?DI;9J?9?:;
C;J78EB?J;I
DL?HEDC;DJ7B ;NFEIKH;I JE 97H87HOB 7D: 9>BEHFOH?<EI  J7?B ' 45
DL?HEDC;DJ7B ;NFEIKH; JE
)!I
.H?D7HO FEBO9O9B?9 7HEC7J?9 >O:HE97H8ED C;J78EB?J;I (!'7 (!)> (!BK 7D: (!)  J7?B '
-% -& 45
(99KF7J?ED7B ;NFEIKH; JE
?ED?I?D= H7:?7J?ED
?7=DEIJ?9 EH J>;H7F;KJ?9 H7:?7J?ED 1 β 7D: γH7OI <79?B?J?;I  J7?B ' 
>;7: '
(-& /,
45
,>E9AM7L; B?J>EJH?FIO <EH
:?IJ7B KH;J;H7B IJED;I
&;D M>E >7: KD:;H=ED; ,0% <EH :?IJ7B 7D: KFF;H KH;J;H IJED;I /, . 45
?=7H;JJ; ICEA?D= ,CEA?D= 9?=7H;JJ;I ED 7 H;=KB7H 87I?I  '
<H7=C;DJ7J?ED
g 45
?=7H;JJ; ICEA?D= J B;7IJ  9?=7H;JJ;I:7O  J7?B '
-% 45
,B;;F :KH7J?ED %ED=;H   >:7O EH I>EHJ;H  >:7O  J7?B ' g 45
%?<;IJOB; <79JEHI 7=?D= O C?9HEDKJH?;DJI ?DJ7A; /?J  /?J  β97HEJ;D; P?D9 7D: <EB7J;  J7?B '
/,  g 45
=?D= 97s;?D; 9EDIKCFJ?ED &;7D 7=;  O H7D=;  O  9KFI 9Es;;:7O  J7?B '
(-& -& 7=?D=
97<<;?D;
9EDIKCFJ?ED
45
7D9;H F7J?;DJI -;IJ?9KB7H 97D9;H 7D: BOCF>EC7  J7?B ' 45
?s;H;DJ 9B?D?97B =HEKFI =;:  O M?J>  <;HJ?B; 7D:  ?D<;HJ?B; <H7=C;DJ;:
7D: DED
<H7=C;DJ;:
IF;HC
?D<;HJ?B; 45
$7HJ7=;D;HI IOD:HEC; $7HJ7=;D;HI IOD:HEC; M?J> <EKH <7?BKH;I E< <;HJ?B?I7J?ED - /, 45
/7H?9E9;B;9JECO :EB;I9;DJI  O -7DD;H IJ7=;I "/ EH / M?J> L7H?9E9;B; =H7:;I "" EH """ /, 45
<<;9J E< L7H?9E9;B; B?D?97BBO :?7=DEI;: L7H?9E9;B; E< =H7:; "" EH """ /, 45
?s;H;DJ 9B?D?97B =HEKFI
!(7I )
IJ>;DEJ;H7JEPEEIF;HC?9 -3 M?J> EH M?J>EKJ L7H?9E9;B; EB?=E7IJ>;DEJ;H7JEPEEIF;HC?9 (-3 EH
87B7D9;: 9>HECEIEC; H;7HH7D=;C;DJI !(  7D:  
 , (-3 -3
7D: -3 M?J>
L7H?9E9;B;
45
.D;NFB7?D;: H;9KHH;DJ
C?I97HH?7=; ?D 9EKFB;I
M?J>EKJ 7 <;C7B; <79JEH
'I; 7I )
,;C;D I7CFB;I <HEC H;9KHH;DJ FH;=D7D9O BEII +)% 9EKFB;I  , +)%
F7J?;DJI
45
<<;9J E< IF;HC DK9B;7H
L79KEB;I
,F;HC 97J;=EH?P;: 8O &EJ?B; ,F;HC (H=7D;BB; &EHF>EBE=O N7C?D7J?ED &,(& H7:;I /, 45
 I?=D?<?97DJ ?D9H;7I;  I?=D?<?97DJ :;9H;7I; g DE ;<<;9J  7J 7D: 78EL;  >;7: '  J7?B ' . 7H8?JH7HO KD?JI  9EC;J ;NJ;DJ - 9EC;J J7?B (-&
(B?L; J7?B CEC;DJ , IF;HC ' <H7=C;DJ7J?ED -& J7?B :?IJH?8KJ;: CEC;DJ -& J7?B ;NJ;DJ CEC;DJ -% J7?B B;D=J> -& J7?B CEC;DJ vs L?IK7B I9EH?D= '
79HOBED?JH?B; -3 7IJ>;DEJ;H7JEPEEIF;HC?9 !( >O:HE=;D F;HEN?:; &,(& &EJ?B; ,F;HC (H=7D;BB; &EHF>EBE=O N7C?D7J?ED (-3 EB?=E7IJ>;DEJ;H7JE
PEEIF;HC?9 )!I FEBO9O9B?9 7HEC7J?9 >O:HE97H8EDI )I FEBO8HEC?D7J;: :?F>;DOB ;J>;HI ) FEI?J?L; 9EDJHEB +)% H;9KHH;DJ FH;=D7D9O BEII
->; ;L7BK7J?ED E< IF;HC 9>HEC7J?D 7D: ' IJHK9JKH; M7I ?D?J?7BBO
KD:;HJ7A;D JE ?CFHEL; EKH KD:;HIJ7D:?D= E< IF;HC7JE=;D;I?I IF;HC
F>OI?EBE=O I;DI?J?L?JO JE H;FHE:K9J?L; JEN?97DJI 7D: H;FHE:K9J?L; 8?EB
E=O 45 ->; ;<<;9JI E< IF;HC ' :7C7=; ED C7B; ?D<;HJ?B?JO
7D: 7II?IJ;: H;FHE:K9J?L; JH;7JC;DJ EKJ9EC;I 7H; IJ?BB 7 C7JJ;H E< :;
87J; 7BJ>EK=> J>;H; ?I IK<q9?;DJ ;L?:;D9; ?D J>; ;N?IJ?D= B?J;H7JKH; JE
9ED9BK:; J>7J IF;HC ' :7C7=; ?I 7IIE9?7J;: M?J> 7 D;=7J?L; ;<<;9J
ED IK99;II<KB 9B?D?97B FH;=D7D9O <EBBEM?D= "/ 7D:EH "," JH;7JC;DJ
45 ->;H;<EH; :?<<;H;DJ IF;HC 9>HEC7J?D 7D: ' J;IJI ?D9BK:
?D= J>; 9EC;J 7II7O >7L; 8;;D KI;: ?D J>; ;L7BK7J?ED E< ?D<;HJ?B; C;D ?D

UNCORRECTED PROOF
G. Gajski et al. Mutation Research-Reviews in Mutation Research xxx (xxxx) 108398
Table 5
->; IF;HC 9EC;J 7II7O <EH J>; ;L7BK7J?ED E< ' :7C7=; ?D >KC7D IF;HC in vitro 7D: <EH 9HOEFH;I;HL7J?ED H;I;7H9>
=;DJIJH;IIEH NFEIKH;I;JJ?D= )7H7C;J;HI J;IJ;: +;IFEDI; +;<;H;D9;I
475) ) ICEA;HI vs DED
ICEA;HI 1H7OI 7I )
475)    7D:  i& )   7D:  i& 1H7OI
 O
 J7?B ' g 475) g )
ICEA;HI 1H7OI
45
&&,    7D:  C&  J7?B ' (-& ↑ ≥  C& 45
);H<BKEHE7BAOB IK8IJ7D9;I ),
!(7I )
);H<BKEHEE9J7D;IKB<ED?9 79?: )(, F;H<BKEHEE9J7DE?9 79?:
)( F;H<BKEHEDED7DE?9 79?: )' 7D: F;H<BKEHE>;N7DE?9
79?: )!N  C& !( i&
 J7?B ' g !( 45
IJHE=;D?9 9ECFEKD:I !(7I ) ?;J>OBIJ?B8E;IJHEB FHE=;IJ;HED; βE;IJH7:?EB DEH7:H;D7B?D;
7D: JH??E:EJOHED?D; 8KJOB7J;: >O:HENO7D?IEB !  i&
!( i&
(-& ;IJHE=;D?9 9ECFEKD:I
!( g !
45
"D>7B7J?ED 7D;IJ>;J?9I >7BEJ>7D; ?IE<BKH7D; I;LErKH7D; 7D: :;I<BKH7D;    7D:
 C&
 J7?B ' >7BEJ>7D; ?IErKH7D;
I;LE<BKH7D; g
:;IrKH7D;
45
ENEHK8?9?D    7D:  i&  >;7: ' -&  i& 45
DJ?C7B7H?7BI !(7I ) HOFJEB;F?D; 7D: ?JI 7D7BE=K;I :?8HECE9HOFJEB;F?D; 
rKEHE9HOFJEB;F?D; 8HECED?JHE9HOFJEB;F?D; 7D: 9>BEHE
D?JHE9HOFJEB;F?D;   7D:  i& !( i&
 J7?B ' (-& g 7DJ?C7B7H?7BI !( 45
EE: 7::?J?L;I ?JH?9 79?:  8;DPE?9 79?:  8H?BB?7DJ 8BK;  IKDI;J
O;BBEM ,2    7D:  i=C%
 J7?B ' -% -&   i=C% 
 i=C% 
 i=C% ,2
 i=C%
45
OF;HC;J>H?D FHEJ;9J?L; HEB; E< /?J
 7D: 
OF;HC;J>H?D  i& /?J   C& /?J   C&  J7?B ' -% -& 9OF;HC;J>H?D /?J 45
'?9EJ?D;   7D:  C& EC;J L7BK;I   C& 45
H?DA?D= M7J;H :?I?D<;9J?ED 8O
FHE:K9JI >7BE79;J?9 79?:I !I
"E:E79;J?9 79?: " 8HECE79;J?9 79?:  9>BEHE79;J?9 79?:
 8KJOB7J;: >O:HEN7D?IEB; ! 7D: 97J7B7I; 7I FHEJ;9JEHI
 J7?B ' (-& !I ! 7D:
97J7B7I;
45
;H?KC :?EN?:; D7DEF7HJ?9B;I !(
7I )
;(')    7D:  C=% !( μ&  J7?B ' 45
-?J7D?KC :?EN?:; D7DEF7HJ?9B;I
8;DP;D; 7I )
-?(')  7D:  μ=% 8;DP;D;  μ%C%  J7?B ' 45
+7:?7J?ED I =7CC7 H7OI    7D:  O J7?B ' -& 45
"Dr7CC7JEHO C;:?7JEHI %;KAE9OJ;I 9EC8?D7J?EDI E< FHE?Dr7CC7JEHO 9OJEA?D;I
?DJ;HB;KA?D 4"%5  "% 7D: "%  "% 879J;H?7B IJH7?DI
Escherichia coli 7D: Bacteroides ureolyticus
% 45
?;J7HO;DL?HEDC;DJ7B 9ECFEKD:I
!(7I )
!;J;HE9O9B?9 7C?D;I EH=7DE9>BEH?D;I )!I CO9EJEN?DI B?F?:
F;HEN?:7J?ED FHE:K9JI 7B9E>EB !(  7D:  i&
 J7?B ' (-& 45
HOEFH;I;HL7J?ED %?GK?: D?JHE=;D <B7I> <H;;P?D= M?J> 7D: M?J>EKJ
9HOEFH;I;HL7J?L; FHE=H7CC78B; <H;;P?D= M?J> 7D: M?J>EKJ
9HOEFH;I;HL7J?L;
% <H;;P?D=
<H;;P?D=  9HOEFH;I;HL7J?L;
45
HOEFH;I;HL7J?ED HOEFH;I;HL7J?ED ?D F7J?;DJI M?J> J;H7JEIF;HC?7 (-& J;H7JEIF;HC?9 I7CFB;I 45
HOEFH;I;HL7J?ED 'EHCEIF;HC?9 7D: <EKH 97J;=EH?;I E< ?D<;HJ?B; C7B;I (-&  J7?B '  >;7:
' 9EC;J DKC8;H ?DJ79J
' DKC8;H % -% -&
J;H7JEIF;HC?9 7D: 7IJ>;DE
J;H7JEPEEIIF;HC?9
45
HOEFH;I;HL7J?ED <H;I> vs 9HOEFH;I;HL;: <;HJ?B?JO IJ7JKI  ,  g 45
HOE:7C7=; FHEJ;9J?L; HEB; E<
79;JOB…97HD?J?D;
H;;P?D=J>7M?D= 79;JOB…97HD?J?D;    CCEB% +  J7?B ' -% (-& 9HOE:7C7=; 79;JOB…
97HD?J?D; 
45
!( FHEJ;9J?L; HEB; E< =7D=B?EI?:;I !( i& JH?I?7BE=7D=B?EI?:;I  -8  i& -% -& !( g  -8 45
HOEFH;I;HL7J?ED FHEJ;9J?L; HEB; E<
=7D=B?EI?:;I
H;;P?D=J>7M?D= CEDEI?7BE=7D=B?EI?:;I  &
JH?I?7BE=7D=B?EI?:;I  -8  i&
 J7?B ' -& 9HOEFH;I;HL7J?ED g
 & -8
45
HOEFH;I;HL7J?ED FHEJ;9J?L; HEB; E<
=7D=B?EI?:;I
H;;P?D=J>7M?D= CEDEI?7BE=7D=B?EI?:;I  &
JH?I?7BE=7D=B?EI?:;I  -8  i&
-& 9HOEFH;I;HL7J?ED g
 & -8
45
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
UNCORRECTED PROOF
G. Gajski et al. Mutation Research-Reviews in Mutation Research xxx (xxxx) 108398
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=;D;H7J?ED7B ;<<;9JI E< =;DEJEN?9 ;NFEIKH;I ?D 7D?C7BI 7D: >KC7DI
Declaration of Competing Interest
->; 7KJ>EHI H;FEHJ DE :;9B7H7J?EDI E< ?DJ;H;IJ
Acknowledgement
->?I MEHA M7I IKFFEHJ;: 8O 7<qB?7J;: ?DIJ?JKJ?EDI 7D: J>; KHEF;7D
EEF;H7J?ED ?D ,9?;D9; 7D: -;9>DEBE=O  (,- 9J?ED 
->; 9EC;J 7II7O 7I 7 >KC7D 8?ECED?JEH?D= JEEB hCOMET

UNCORRECTED PROOF
G. Gajski et al. Mutation Research-Reviews in Mutation Research xxx (xxxx) 108398
References
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45 & &KH7JEH? % -7C8KHH?DE , &7H9>?7D? & 7C8?  (B?L?JE  PP7H?
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7FEFJEI?I ?CC7JKH?JO 7D: EN?:7J?L; IJH;II &EB &;:   
>JJFI:E?EH=CEBC;:
45 + !;DA;B $ %;?I;=7D= (H?=?DI E< IF;HC ' :7C7=; &7B; "D<;HJ?B ,FH?D=;H
"DJ;HD7J?ED7B )K8B?I>?D= >7C  FF  >JJFI:E?EH=
6
45 - +:E , !7C?BJED &( IIKCFYWE ,F;HC ' <H7=C;DJ7J?ED 97KI;I
7D: ?:;DJ?q97J?ED 3O=EJ;    >JJFI:E?EH=

UNCORRECTED PROOF
G. Gajski et al. Mutation Research-Reviews in Mutation Research xxx (xxxx) 108398
,
45 #( %?DI9>EEJ;D ' /;H>E<IJ7: $ KJPAEM  (BI;D  27KA 2 (B?=I9>BV=;H
HKD8EH= # ,9>EEJ;D +0% E:I9>7BA )7J;HD7B B?<;IJOB; 7I 7 FEJ;DJ?7B
IEKH9; E< =;HCB?D; CKJ7J?EDI JH7DIC?JJ;: JE E<<IFH?D= , #  
 >JJFI:E?EH=<@
45 . $;BBD;H ) +K:EBF> + )7HM7H;I9> !KC7D '-EFE?IEC;H7I;I
:?7=DEIJ?9 7D: J>;H7F;KJ?9 ?CFB?97J?EDI <EH 7D9;H (D9EB +;I -H;7J  
 >JJFI:E?EH=
45 +& %78;H=; E?IIEDD;7KBJ (D J>; D7JKH; 7D: EH?=?D E< ' IJH7D: 8H;7AI
?D ;BED=7J?D= ,F;HC7J?:I ?EB +;FHE:    >JJFI:E?EH=
8?EBH;FHE:
45 +# ?JA;D + HEDIED - ,C?J> ' ; "KB??I ->; IEKH9; 7D: I?=D?<?97D9;
E< ' :7C7=; ?D >KC7D IF;HC7JEPE7 7 9ECC;DJ7HO ED :?7=DEIJ?9 IJH7J;=?;I
7D: IJH7M C7D <7BB79?;I &EB !KC +;FHE:    >JJFI:E?EH=
CEB;>H=7J
45 +# ?JA;D ' ; "KB??I (D J>; FEII?8B; EH?=?DI E< ' :7C7=; ?D >KC7D
IF;HC7JEPE7 &EB !KC +;FHE:    >JJFI:E?EH=
CEB;>H=7F
45 # HEM ->; EH?=?DI F7JJ;HDI 7D: ?CFB?97J?EDI E< >KC7D IFEDJ7D;EKI
CKJ7J?ED '7J +;L ;D;J   >JJFI:E?EH=
45  EH?;BO (& 0?BA?; )7J;HD7B 7=; ;<<;9J CKJ7J?EDI 7D: I;B<?I>
IF;HC7JE=ED?7B I;B;9J?ED 97KI;I 7D: 9EDI;GK;D9;I <EH >KC7D :?I;7I; C #
!KC ;D;J   >JJFI:E?EH=@7@>=
45 ') ,?D=>  7DD;H ++ -?9; &- &9EO  EBB?DI % ,9>D;?:;H
8KD:7DJ 7BA7B?I;DI?J?L; I?J;I ?D ' E< >KC7D 7D: CEKI; IF;HC NF ;BB +;I
   >JJFI:E?EH=
45 # %;; &# BB;D + 7B>EHD JEC?9 <EH9; C?9HEI9EF; 7D7BOI?I E< 9>HEC7J?D
LEBKC;I ?D >KC7D IF;HC M?J> >;7:I>7F; 8DEHC7B?J?;I ?EB +;FHE: 
  >JJFI:E?EH=8?EBH;FHE:
45 & (BB;HE  ?B KPC7D & %EF;P +$ ,>7HC7  =7HM7B $ %7HIED 
L;DIED # ->EC7I #  BL7H;P >7H79J;H?P7J?ED E< IK8I;JI E< >KC7D
IF;HC7JEPE7 7J :?s;H;DJ IJ7=;I E< C7JKH7J?ED ?CFB?97J?EDI ?D J>; :?7=DEI?I 7D:
JH;7JC;DJ E< C7B; ?D<;HJ?B?JO !KC +;FHE:    >JJFI
:E?EH=>KCH;F
45 # #7DI;D $ (BI;D + 0?=;H ! '7;=;B? ) :; E;H  L7D ;H !E;L;D #
!EBC; HKD8EH= % &KBB;D:;HI 'K9B;EJ?:; ;N9?I?ED H;F7?H ?D H7J C7B; =;HC
9;BBI BEM B;L;B E< H;F7?H ?D ?DJ79J 9;BBI 9EDJH7IJI M?J> >?=> :K7B ?D9?I?ED 79J?L?JO ?D
L?JHE 'K9B;?9 9?:I +;I    >JJFI:E?EH=D7H

45 # ,>7C7D 2 27C7K9>? 0, 07H: ,F;HC ' <H7=C;DJ7J?ED 7M7A;D?D=
J>; IB;;F?D= =;DEC; ?E9>;C ,E9 -H7DI    >JJFI:E?EH=
,-
45 2 27C7K9>? # ,>7C7D ,& E7P 0, 07H: )7J;HD7B FHEDK9B;7H '
:;=H7:7J?ED ?I <KD9J?ED7BBO B?DA;: JE ' H;FB?97J?ED ?D CEKI; (E9OJ;I ?EB
+;FHE:    >JJFI:E?EH=8?EBH;FHE:
45 + ,7B;>  =7HM7B  '7:7 &! B-EDIO +$ ,>7HC7  &;O;H +
';BIED # ->EC7I ';=7J?L; ;<<;9JI E< ?D9H;7I;: IF;HC ' :7C7=; ?D H;B7J?ED
JE I;C?D7B EN?:7J?L; IJH;II ?D C;D M?J> ?:?EF7J>?9 7D: C7B; <79JEH ?D<;HJ?B?JO
;HJ?B ,J;H?B  ,KFFB    >JJFI:E?EH=I

45 ,& %;M?I " =87@; # BL7H;P ,F;HC ' J;IJI 7I KI;<KB 7:@KD9JI JE I;C;D
7D7BOI?I ,OIJ ?EB +;FHE: &;:    >JJFI:E?EH=

45  K? + ,>7HC7 + !;DA;B  =7HM7B +;79J?L; ENO=;D IF;9?;I ?CF79J ED
IF;HC ' 7D: ?JI HEB; ?D C7B; ?D<;HJ?B?JO D:HEBE=?7   ; >JJFI
:E?EH=7D:
45 +# ?JA;D % +O7D # KHHO & 7A;H &KBJ?FB; <EHCI E< H;:EN 79J?L?JO
?D FEFKB7J?EDI E< >KC7D IF;HC7JEPE7 &EB !KC +;FHE:   
>JJFI:E?EH=CEB;>H=7=
45 , KD;I & B,7:77D  =7HM7B ,F;HC7JE=;D;I?I ' :7C7=; 7D: '
H;F7?H C;9>7D?ICI ?D C7B; ?D<;HJ?B?JO +;FHE: ?EC;: (DB?D;  
 >JJFI:E?EH=@H8CE
45 #( %?DI9>EEJ;D # %7K8;DJ>7B  ;C;B?  7KC=7HJD;H  D:;HIED /
,?F?D;D HKD8EH= +&& !7;D;D  J>;DEK ## H?;:Z # L7D
,9>EEJ;D +0% E:I9>7BA "D9ECFB;J; FHEJ;9J?ED E< =;D;J?9 ?DJ;=H?JO E< C7JKH;
IF;HC7JEPE7 7=7?DIJ EN?:7J?L; IJH;II +;FHE: -EN?9EB   
>JJFI:E?EH=#+)+(-(1
45 ++ !;DA;B + H7DA;D ,F;HC ' <H7=C;DJ7J?ED EH?=?D 7D: ?CF79J ED
>KC7D H;FHE:K9J?ED # +;FHE: ,J;C ;BB ?EJ;9>DEB    >JJFI
:E?EH=
45  EDPTB;P&7H\D # EITBL;P + +EO -OF;I 97KI;I :;J;9J?ED 7D: H;F7?H E<
:D7 <H7=C;DJ7J?ED ?D 7D?C7B 7D: >KC7D IF;HC 9;BBI "DJ # &EB ,9?  
 >JJFI:E?EH="#&,
45 # +?87I&7ODEK # ;D;J ,?D=B; 7D: :EK8B; IJH7D: IF;HC ' :7C7=;
:?s;H;DJ H;FHE:K9J?L; ;<<;9JI ED C7B; <;HJ?B?JO ;D;I 7I;B   
>JJFI:E?EH==;D;I
45 !# >? 2 >KD= ,2 >E? #! $?C 2 $?C #, %;; !, %;; &
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IF;HC7JEPE7 9ECF7H?IED M?J> IEC7J?9 9;BBI ?EB +;FHE:   
>JJFI:E?EH=8?EBH;FHE:
45 K;DJ;I&7I9EHHE ! ,;HH7DE  +EI7:E ,F;HC 9>HEC7J?D H9> D:HEB
   >JJFI:E?EH=

UNCORRECTED PROOF
G. Gajski et al. Mutation Research-Reviews in Mutation Research xxx (xxxx) 108398
45 & 7H:?D;H 7H:;D & 7BB;IJ;HEI & EH:ED )) -7C !?IJED; 7D:
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=BE8?D :EC7?D ?D >KC7D IF;HC &EB ;BB ?EB    >JJFI
:E?EH=C98
45 # +?87I&7ODEK  7H9?7ED7L?B7 ( !?:7B=E # 7J7BTD # &?H^ &
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;KJ>;H?7D C7CC7BI KD:;HB?; :?IJ?D9J BOI?I H;GK?H;C;DJI HEDJ ;BB ;L ?EB 
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=BO9?:7C?:;?D:K9;: ' B;I?EDI ?D CEKI; C7B; =;HC 9;BBI 7D: ?D CEKI; 7D:
>KC7D BOCF>E9OJ;I &KJ7J +;I -EN?9EB DL?HED &KJ7=;D   
>JJFI:E?EH=#&+ '-(1
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) /?BB7D? +;)HEEC;J  ';M "D /?JHE &;J>E: JE II;II ' 7C7=; ?D
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JENI9?A<C
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EC;J 7II7O <EH 7II;II?D= ' :7C7=; ?D IF;HC7JEPE7 +;FHE: ?EC;: (DB?D;
   >JJFI:E?EH=IN
45 " EHJ;I KJ?;HH;P  %EF;P;HD7D:;P #% ;HD7kD:;P &" 7L?B7
+E:H?=K;P , #E>DIJED # EI7BL;P "DJ;HFH;J?D= IF;HC ' :7C7=; ?D 7
:?L;HI; H7D=; E< C7CC7B?7D IF;HC 8O C;7DI E< J>; JMEJ7?B;: 9EC;J 7II7O HEDJ
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E:I9>7BA # /7D ,9>EEJ;D  D:;HIED HKD8EH= "D L?JHE ;L7BK7J?ED E<
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&KJ7=;D;I?I    >JJFI:E?EH=CKJ7=;=;G
45 ,& JJ?7 , >C7: +& (A7I> , 7A>;;J HE9BEH ?D:K9;:
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' IJH7D: 8H;7AI 7D: EN?:?I;: 87I;I ?D IEC7J?9 7D: IF;HC7JE=;D?9 9;BBI <HEC
J>; ;7HJ>MEHC ?I;D?7 <;J?:7 7<J;H ;NFEIKH; JE ?ED?I?D= H7:?7J?ED &KJ7=;D;I?I 
  >JJFI:E?EH=CKJ7=;=;H
45  ;C;B?  D:;HIED &;9>7D?IJ?9 ?DL;IJ?=7J?ED E< +(,"D:K9;: ' :7C7=;
8O E;IJHE=;D?9 9ECFEKD:I ?D BOCF>E9OJ;I 7D: IF;HC KI?D= J>; 9EC;J 7II7O "DJ
# &EB ,9?    >JJFI:E?EH=?@CI
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>JJFI:E?EH=@J>;H?E=;DEBE=O
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>JJFI:E?EH=@C7HFEB8KB
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:E?EH=@7GK7JEN
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9;BBJOF; I;DI?J?L?J?;I &KJ7J +;I ;D;J -EN?9EB DL?HED &KJ7=;D  
 >JJFI:E?EH=@CH=;DJEN
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2 %? -EN?9?JO E< 7HI;D?J; JE ;7HJ>MEHCI 7D: IK8I;GK;DJ ;<<;9JI ED IE?B FHEF;HJ?;I
,E?B ?EB ?E9>;C    >JJFI:E?EH=
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