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Abstract

Stable suspensions of NiO-NPs and Mn3O4-NPs nanoparticles (NPs) with a mean (±s.d.) diameter of 16.7 ± 8.2 nm and 18.4 ± 5.4nm, respectively, purposefully prepared by laser ablation of 99.99% pure metals in de-ionized water, were repeatedly injected intraperitoneally to rats at a dose of 2.5 mg/kg 3 times a week up to 18 injections, either alone or in combination. A group of rats was so injected with this combination with the background oral administration of a “bio-protective complex” (BPC) comprising pectin, vitamins A, C, E, glutamate, glycine, N-acetylcysteine, selenium, iodide and omega-3 PUFA, ths composition having been chosen based on mechanistic considerations and previous experience. After the termination of injections, many functional and biochemical indices and histopathological features (with morphometric assessment) of the liver, spleen, kidneys and brain were evaluated for signs of toxicity. The accumulation of Ni and Mn in these organs was measured with the help of the atomic emission and electron paramagnetic resonance spectroscopies. From the same rats we obtained nucleated blood cells for performing the RAPD (Random Amplified Polymorphic DNA) test. Although both metallic NPs proved adversely bio-active in many respects considered in this study, Mn3O4-NPs were somewhat more noxious than NiO-NPs as concerns most of the non-specific toxicity manifestations. Moreover, they induced significant damage to neurons in caudate nucleus and hippocampus, which may be considered an experimental correlate of the manganese-induced Parkinsonism. The combined toxicity of these NPs is of a complicated type (additivity, synergism or subadditivity) depending on the effect assessed. The comparative solubility of the Mn3O4-NPs and NiO-NPs in a biological medium was measured in vitro and discussed as one of the factors underlying the distinctions between both their toxicokinetics and their toxicities. The BPC tested by us attenuated both the organ-systemic toxicity and the in vivo genotoxicity of Mn3O4-NPs in combination with NiO-NPs. Keywords: nanoparticles, manganese (II,III) oxide; nickel (II) oxide; subchronic toxicity; bioprotectors.
Comparative experimental modeling of subchronic toxicity of
nickel (II) oxide and manganese (II,III) oxide nanoparticles
and the attenuation of their combined adverse effects with a
complex of bioprotectors.

 ! "#  "#$
%&'$(')(#'
#!  '$
1The Medical Research Center for Prophylaxis and Health Protection in Industrial
Workers; 30 Popov Str., Ekaterinburg, 620014, Russia
2 The Institute of Natural Sciences, the Ural Federal University, Ekaterinburg 620000,
Russia
3 The Ural State Medical University; 17 Klyuchevskaya Str., Ekaterinburg 620109,
Russia
4Institute of Industrial Ecology, the Urals Branch of the Russian Academy of Sciences,
20 Sofia Kovalevskaya Str. ,Ekaterinburg 620990, Russia
*+ $,+#-.",,(/0
,,12$($2"3$2,(2"045,,12$($2$1121(,06127""2".2$,27,028
-90-9'
Abstract
- + 4 )%2)  $%(2) + :); < 
:=;4.1=>">(=3(+'++4'++
-'-47777?+2<<+'@
+'4"3A$<+>@ 
-+4<@<  -<  
-4B-2++5C:*;++
*# ')2'' 2$
DE + - - 
1
+ 5+ 4    4 @ ' 4 
-  + 4:< + ;4
   + '  - <  4  4 5' F 
4) <<   +4 
+++E <
-  -  4 +4   /G :/ +4
'+ G);H
  - )+'-2'+
 '$%(2)<< 5 )%2)4
  2+45'4 '4
     +++ <  ' -  
5+4 2F -5'
4 )4+'+:''-';+
 44F +-'4 $%(2))%2)
-<in vitro 4 4'
 -<-  5 5F *-'
-  2' 5'  in vivo 5' 4$%(2
)-< )%2)
Keywords: nanoparticles, manganese (II,III) oxide; nickel (II) oxide; subchronic
toxicity; bioprotectors.
1. Introduction
)+:);454+45'
4+  '- 5
-4++4'4:;)4 --
 '  - ++ <    +  - 4  
   -' 2<   
    ' <   '
4  +  -    I,,   
4:J,,;
2
F +@' 4  +-    '5' 
5'4)-''-')-
in vitro5+4-4+'4
+'   4 5' &< ' 5+ 4  5+
        < - 4  
+   + + 4 5' : +K
5 2+  +    '    
444'4+ ;'-'
  5+< 
L @  5+  )
5- in vivo '+<  
-+<  +4'5'
+within 2M+5
 4 2  + -<    -
4+4+  +5 
     -      :   ",, ", ",30
",(;
 < -'  - :2
< ''+';'+'+- 
+5 ++++45
4       
+4  )M  +-<  '-'
++4 '+ +4 
+  % 4    4 4  5' 
 4   4M   '   ' + -  +
4 545)<4 -
44'+ 44'5' 
'<   4 '544+-'
)<  ''-' -
 : <-'EN  ",$;4 4
5'+4-<   
+4 55'4
   +: -4<;
'4<+M4 445 -'
3
 '44)+ - 5+in vivo
4 +  +  L  '   
4)4:",$;&<<+
- )'' +  
- -   -  <  ' M   ' < 
 4+' +8 <  
24 + 4 any  +   -' 
  4  )24 - F  4  '   
-     '  4      
  ++4 544+-'5) 
'+ 
  54   <    )4  5
<  4+ -  <+-42
4<4)'+ 44:;5
:)%2);-45' +4<  
:;5:$%(2); :",,0F-","0@
",(;#5+ 4   5'4  2 2
)        
-+-'   : O   77> ",,$ ' 
O ","",*+",(- ",,.  
",$",(;-4<<+ 
-+- 5+in vivo
F 5++45 44
  +  -5' 
4 4-4 -+ 
44:*7>770/*77,;- 54
 < )P"  %(   <   -' 44 '+ 4
-5' : 4  '+ +4 +'<
-';+ 4444:
",3;
 ++< +-'+4
 5' L  +'  5'4)
1 & 2 Q +5'
< '-+ 4'-
++4- <<R
4
< <  4+-5+++
  44 <   +4 -4 -  4
<     +' 44  F   + 4 
B- ++ '5C 4 44 +   5 
  +   ++  <  5+ 4 
+   - +-  -'     +'
<:",,>",(;<-5+
 +4 +' 445'<
%+5++ - '5' in vivo 5'4
 :",$;4++5:",(;)<
'  < -   4 2+
-+ +5 :*; F  * + + 2
++ 2$ DE-   
+<444- 
5'L 4 : ",(;     < -
 ' )2' '   2 
++ '  ( < before     4 )%2
)6$%(2)+:,"3  ;  4'<
+ ' <<' 5+< 
'+ )<<  -   +44' 4 -2
++5'-< ++@4
-4)
2. Materials and methods
F 5+< -< 4 4<-
'<  -'< 43,"",< "44
5+ +/ <   - 
4<4-4F 5+<+
+   <   B  ++ 4 -
 C+-' *4%4
  :7>3;  < ++ -'   #  * 4  
#-/ *4+ '5& 
L
5
E  5+<++ +4 5+ -'
-4+<+4+< 
4 777?< +  - 4  <' <  <
-<+4E2",/+'
: F '*-/;-'-2++
4-:+  ,,+  "&< ,.( ;F 
''<->,SA"F <<  
4 +"1,A -'<
4++4 4
 + :#; * L  :* O
'; <  4    4   + / 4
+ +  $,, / :LF '; <  4 ' 4   )
+
F <,3 A -'+ + 4 +'
+43 3,T*F +- +<4+ 
 +:E;F   + <-'/ ++'
:E "  $; F   +  :=G; - -' 
+4 4+':#;<.1=>"
4)%2) >(= 3(4 $%(2) F - 4 <
'- :E"-$-;
F  - 4 -       +  <      + 
+ 4   +  + $, ' 4 + ++
4   -' 4  )%2)+ F  $%(2) +
<--4+<' -' 2
@+ @) -< 4
 +
6
E#4)%2):4;$%(2): ;+
+--'+'
:; (-;
E"* 45 ) + -'  -
:;H/ + :-;  - 4 - -' 
+4#)
:; :-;
E $ *  4  5 + + -'
-:;/+:-;-4--'
+4#4)
#   ++< +':;$
<:+>@;4,3+:-"3A;
4   +F  -< ' 44 )
7
    4    )%2)  $%(2) + <
<  44 '  @ +'     4   -
5++4 4-F 4 +
5+)4 )%2)$%(2)<'@<
 4  <:4  -  4++ 
+;-' <   4 + '
@4 <
++4<@<  4:)%2)6$%(2);
)-< -4-2+ +5:*; -
-< +< *+@4<
' 4    4   5+ +   4< + <
+448
L 
#4 *)-'4+4-2  +
U 4  < <45  4-' + 

/4 -4 2+  4 2-:<  
+-4545+''4M' 4'
 - 4445;
* 4 '  4 ' 4  + :; +4 '
:';+ ++ 'V2 :V2; 
)
+4+'-4  45  
-   4 '   4 
 ' :G&; '  '+ ' :-'    4
4 -44<  
+';
F     <  -' +  - <  -'
5 F   + '  - < <  F 
-    4   -    
:&;   + - - -- +
8
' ' :G;  + +  2  +2
:FF; ' 4&2+
     '+  4 '++ '  '5 
 '
  -2-'  - <+4
<2< M-'W:F773;X
F 44 44-< + 
<Rt< 44++
-  - 4 -<  +4' 4  + +<   
 +4' <  +  + 5+)%2) 6
$%(2)6 *<  5+ )%2)$%(2)<  
*0 <+ +5+ ):+'
-;<   *< ')
F 4 :); +4 '
+-<-' ++':#; 
+< '++*.3,,G
:F  4 D; + 4   42   <
-@ <   + 4/3 <
'F )%2)$%(2))4 +4 
<-' +:#/; 
+  + #Y  : '; F 
4 42 <+
+'- <++4 (4
+4 5-'  5
< ')RE+  4
 < R+ 
+'*:%'+';
Testing of the in vivo genotoxicity with the Random Amplification of Polymorphic DNA
(RAPD) Test on blood nucleated cells.
F'<'..-+ + +F +
<   +    Z>, TP F  <   ++'
'2+-'FG)4 
9
< # :; 4  <   4R
 4  F  G)  4   + < 
++ ':D+,,+    
D;0  '<4 Z>(TP:'# *
 S+; -4 /G:/+4'+ 
G);+4--':",,1;F  
-    4    4 G) <       
+ 4 224G) '<
4'' +: ; 4
-' 4G)4G)+4
< +4+   F   
4 G) <  B444 4C  4
'44 4  
Choice of bioprotectors
 <   454)
 5+    4  4  -+   
5:-'",(; -'2
:",$;  2*% : ",(;<   
4< - 4    +- +    
-- 5< )%2)$%(2)8
:;44--   4
4F' 5+ 4'5+
:7>"0",,1",,7",$;
   4 + 4 <  +<4 
+  5  - 4  '  4
' )R '5' 5' :EN   ",$; 
 2+4-2+444<-
  +4'5'
  R @    5'     
  '       + 4  -
4    45''-
<' + 5+4
4     +  ' :   ",$0 '2
L'<  ",$; L 4++   
10
++'     < +' + 4    44 4 
5'
"F   < +8 '' :   '
  -' < - 4 4 )2'';  
 -    +  +' -'   5  
 4)'5' 5+  
 4'+5' -
:G771;
$% +4 R25':[\P
;
(]2$+'4'<  
 G) +    +'  + +   + <  <
 -'    5+   5  :
:",,1",$0",(;;
3+5-4  '4
-'5: ",,1;
.-     +  2-+ 45
5     < - :<     4 + + 4
5+'-' ;
The doses and administration methods of these bio-protectors are given in Table 1.
Table 1.
Doses and the mode of administration of the bioprotectors tested in our
experiment
Bioprotectors Estimated dosage and the mode of
administration
+++ 1 g/kg (added to the fodder)
 160 mg per rat (as a 1.5% drink instead of water)
' 12 mg per rat (added to the fodder)
)2'' 30 mg per rat (added to the fodder)
*
4.4 mg per rat (added to the fodder)
#
11
0.84 mg per rat (added to the fodder)

4.0 mcg per rat (added to the fodder)
*4  
$ DE 1 drop per rat (sublingually)
Potassium iodide
4.0 mcg per rat (added to the fodder)
L 3?4<ad libitumF 
B-G<C:-' #2  O '/; 4 ++ 
DE'4 ]2$+:"(?;<  4
+F +++-:-'/;
<  R 4 M'+  ,,, A
-'< *++4-
- <        + 4   4  M
+'4 4:< 
 <<'4  R
M<- +R-;
F        - 4 +-'   
M4< 4 -2
 - - <       <  
 4) 5' )   - 
<     + <    < '4-
44F < '- 5++4<
 *-<5+'5
2. Results
  -  4 F- "    )  :  -' #; <
4'   5+  - )%2)  )%2)6$%(2)
+ +'#5+$%(2)@:
-< )%2); -    4 
'-' 4) 4 
 -<4'  +<  5+)%2)
F <+ +< -*
 )4  ++"<:J,,3;+
12
<   +5+ )4 )%2)6$%(2)<  *D  
44 * 4  + 
-4'<  * 
'
Table 2
Total manganese and nickel contents of rat’s organs, mcg per g of dried tissue,
after repeated intraperitoneal injections of NiO-NPs or Mn3O4-NPsnanoparticles,
together or separately,and of their combination with background oral
administration of the BPC (X±s.e.)
Metal
GROUPS OF RATS given :
Control NiO-NPs Mn3O4-NPs NiO-
NPs+Mn3O4-
NPs
NiO-
NPs+Mn3O4-
NPs
and BPC
Liver
Manganese 1$$=,3. .$$=,(7 1",=,1$ .>,=,", 11=,$
Nickel $1=,.3 .(,=$".^6_ ">,=,",6$>,,=">,^ >3,=3.^6
Spleen
Manganese ">>,=1( $",,=(3 "3,,=>1 "3>$=("$ (,,="$$^6
Nickel "3.,=>7 (.13=>((^ $"3,=(.$ $.1=1" $."3=>71
Kidneys
Manganese $13=,73 ,,,=,.$^_",3,=(3^6,>,= .1=."6
Nickel >",=3. ",.,=$"$ .,,=3> 1.,="7 >.1=">
Brain
Manganese .,,=,(3 11=,7> ,13=1,^ 1.,=,> 7",=,>,^
Nickel (.1=,7. ">$=", 313="(. 3>,=".3 >.,=,.,6
13
Note:*. statistically significant difference from the control group; +. from the group given
NiO-NPs+Mn3O4-NPs (without the BPC); from the group given Mn3O4-NPs (p < 0.05 by
Student’s t-test)
Table 3
Total manganese and nickel renal excretion in rats after repeated intraperitoneal
injections of NiO-NPs or Mn3O4-NPs nanoparticles, together or separately, and of
their combination with background oral administration of the BPC, mcg per 24 hr)
(X±s.e.)
Metal GROUPS OF RATS given:
Control NiO-NPs Mn3O4-
NPs
NiO-
NPs+Mn3O4-
NPs
NiO-
NPs+Mn3O4-
NPs
and BPC
Manganese ,,.=,,3 ,,,>=,,,.6_ ,,.=,,6,3"=,^ ,">=,
Nickel "7=," $17=3$^6_ ,.$=,$^6.1="7^ (,>=."^6
Note:*. statistically significant difference from the control group; + from the group given
NiO-NPs+Mn3O4-NPs (without the BPC); from the group given Mn3O4-NPs; (p < 0.05 by
Student’s t-test )
 )%2)$%(2): +);<<   +
4#/++' +'<  #/4  $%(2
)< < 4M4  4  )%2):E (; <
<  <       :    ) ;    -
5+ ++<  +5+)%2) 
<-'*
  -  4 F- $     5+  )%2)   ' 
5 4)<'4'F + 
 5+  )%2)6$%(2)< 4 - 4  +
<   5+ )%2) ':J,,3;<     '5+ 
)%2)6$%(2)< *444 4
14
  )%2)25+       5+  $%(2)
5'< -< 5+)%2)- 
<4  *
E( ): )%2);4    +   +4
5+  )%2)  $%(2)6)%2) - 
<   *:  )%2) 6)  6* +'; #/
 G+ 0-73?*
Table 4
Some functional indices for the condition of rat after repeated
intraperitoneal injections of NiO-NPs or Mn3O4-NPs nanoparticles
separately and of their combination with background oral
administration of the BPC (X±s.e.)
Index Control Mn3O4-NPs NiO-NPs NiO-NPs+
Mn3O4-NPs
NiO-NPs+
Mn3O4-NPs
and BPC
BPC
-'
73,=(> ",1$=(7 ",,=$, ",$"=$, ",,,=37 ",(3=3(
E-' "$"3=33 "($"=(. "((,="7 "(1$=.. "$>.=1. "$73=33
'? 7(=" 13=( "3=$ ".=", 7(="$ 1$=(
+
,,-' $3=, (>=,^6(,=,^_$7=, $1=," $1=,"
15
++
,,-' ,$.=,, ,(>=,,$^ ,(=,," ,((=,,$^ ,$1=,,$ ,$>=,,"
'+
,,-' ,.(=,, ,."=,,"^ ,37=,,^ ,37=,,^ ,3>=,, ,.(=,,"
+
,,-' ,1.=,, ,1"=,,^ ,1"=,," ,1=,," ,.>=,," ,11=,,"
F+
4-2
  +

37=,. 1"=,$ 1.=,$ .1=, .7=,7 .7=
)-4 2
+ 
$
7"=,7 "$=,.^63$=^_.,=,^ $$=(^ 3(=3
&-A 1,=," .3=," .$=,^ .,=,(^ .3=," .7=,
#' ',"
A $(=, "7=,^ "7=,^ ">=,"^ $=, $"=,
4
-`$.3=, .37=,^ .(.=,^ .(7=,^ .>=,76.37=,>^
&? "$=,$ 7"=,.^ >7=,3^ >$=,3^ 71=,3 ",1=,3
/'a "(=$ "3"=$1 $=$^ "1$=$, "3="$ 13="$
',$A` ($=,( 3"=,$ ("=,(6.=,3^ 31=,.6($=,(
F -'
-,$A` $.1.=>> $>3>=",3 $37=>( (,$=3 $>>"=77 $131=>,
'+ '? 7$1=, >(3=",^6>3.="$^ 7,3=3 7$=" 7$"=,>
'? (7=,7 ""=.^6=",^ 11=$ 1"=,7 3(=,1
'? $=," $$=,.^6$"=,(^6>=,$ .=," (=,$
--
bA ","=,( ,71=,^ 3=,^ 3=,^ 3=,61=,

 '
:G&;'
-44
+3,
'+ '
.(1=(3 33.=",.^ 3.,1=3^ 3$,"=""3^ 3.7=1. 37,=3
^
F+
4-A 1.,=$ 13"=161"=1^ .7(=7^ 1"1=" 17(=3
16
-4
-A (..=,> $>$=,7^ ("3=^6_ $>.=,>^ (>=6(1$="
-4-
A "7(=,7 $.7=3^6">>=._$,1=( $,7=,7 $"="
A5 .=,,. =,,3^63=,6_ $=,,(^ (=, 3=,
F'-
A ^ $1>3="31 "13,="3"^ "17(="(.^ "17,=>(^ $$>>=($3 $,3>=>3
^
F'-
A ^ 13>=(> 3(1=(1^ 333=$$^ 3,3=$(^ ."$=.$ .,="7^
G/44 3,=," 3,(=," (71=," 3.>=,( 3$1=,$ 3,7=,$
&2+-
`A (7=,3 >,=( 31=,7 ..=> "7=1 (7=$
D-
`A 31,=> ..=""^ "3=7^ 7$=17^ ($$=37 "(=.1^
D-
A 3(=,3 3,1=,. (7=,1 (.=,3 3$=,3 3>=,3
'4c2
4
-
A:^;
,.=,7 >>=,> 7$=$ >=,3^ 7,3=," ".=$
*-
`A (>(=7 ((.=,7 (.(=,7 (($=3 (("= (>$=$
+ + 
-
A:^;
,1=17 $$=>7 "(>=(( 3"=." 37=>1 ,($=,7
*-
`A .=, ,7=,"^ =,^ ,7.=,"^ $3=, .=,
/ 
 - '
`A
"".=7 >(= .>=.^ ""=". ",3= 1>=1
*+
-A? 1"="7 7>$=.">^ ,,>=.$^ ,$=3,^ ".=7. 13.=(3
G-
A 3"=, (3=,"^ (7=," (33=,"^ ((7=," (7"=,$
F '+ 
4 '++ '
-d\A
3=," "=," 1=,( 3=,$ ,7.=,( ,=,"
17
F '5-
+A .1=,. $>=,$^6(>=,3^ (1=,(^ (.=,( (>=,"^
F '
-+A ">=,7 (>=. $1=, $1=,1 $"=,1 "=,.
G $"1=> $,,=$6$(1=$3617="7^ $,"="16$"=(3
D'
,1
=,,,
,1
=,,,"6
,.
=,,,6
,"$
=,,,^
,7
=,,,6
,7
=,,,
A ,$=,," ,3=,, ,=,,6_ ,7=,,( ,.=,,$ ,$=,,
D
A (.>=>( 3(.=>7 ($$=7 >$.=3$ (1(=>( $("=1.
D
`A 7>=>7 $>$=(^ 7>(=.,6_ ..,=7^ ..=7 >7>=."
*
A ,7=, (=,^ $=,,>6 >=,"^ "=,6"=,
#

A
,1.=,,$ ,7,=,,$^6 ,>(=,,3 ,..=,,1 ,1>=,,( ,1"=,,3
VQ
`A .1=. >"1=> 7=> 7,=3 ."=,. >(=$
VQ
`A' ,"$=,,1 ,"7=,,3 ,$(=,,> ,3(=,$ ,""=,,"6,"3=,,>
Note:*. statistically significant difference from the control group; + from the group given NiO-
NPs+Mn3O4-NPs (without the BPC); from the group given Mn3O4-NPs (p < 0.05 by Student’s t-test with
Bonferroni correction).
Table 5
Morphometric indices for tubular epithelium damage in the kidneys of
rats after repeated intraperitoneal injections of NiO-NPs or Mn3O4-NPs
nanoparticles, together or separately, and of their combination with
background oral administration of the BPC (X±s.e.)
18
GROUPS OF
RATS GIVEN
BRUSH BORDER LOSS
(% LENGTHWISE)
EPITHELIAL DESQUAMATION
(% LENGTHWISE)
Water (control) 3((=,7 ,,,=,
NiO-NPs ,$=1^ ,(>=,$.
Mn3O4-NPs 7,"=1^ ,">=,$"6
)%2)6$%(2) "$$="$^ "($=,^
)%2)6$%(2)6
*
1,>=1 ,,,=,6
Note:*. statistically significant difference from the control group; + from the group
given NiO-NPs+Mn3O4-NPs (without the BPC); p < 0.05 (p < 0.05 by Student’s t-test with
Bonferroni correction).
Table 6
Morphometric indices for the state of rat’s liver and spleen after repeated
intraperitoneal injections of NiO-NPs or Mn3O4-NPs nanoparticles, together or
separately, and of their combination with background oral administraon of the BPC
(X±s.e.)
INDICES GROUPS OF RATS
Contr
ol
NiO-NPs Mn3O4-
NPs
NiO-
NPs+Mn3O4-
NPs
NiO-
NPs+Mn3O4-
NPs
+ BPC
LIVER
Akaryotic
hepatocytes
per 100 liver
cells
"1$=,">
17$=,3^6 .,$=,("^
3($=,(^$$>=,$(6
Binucleated
hepatocytes
per 100 liver
cells
13=,..
($$=,.$6 >,$=,.,
>(,=,7" .",=,1"
19
Kupffer cells
per 100 liver
cells
"3=,33
,",=,.$6 "13=,(3^6
.$,=""^""=,16
SPLEEN
Red pulp to
white pulp
ratio
>$=( (3=,7^ 3"=$ $.=,(^ ."=$6
Number of
brown
pigment
micro
aggregates
per square
of the
Avtandilov’
s grid
, "3=,"_.(=,$^6,(=,(^ 37=,"^6
%4 3,4- 4 R
F-('44 <-
""4 444)%2)".4 444$%(2)"34 
-  4    )   '   5+    
-<    - *    '4 
<    <  55+   4.
:F-(; 4-'<25+<
&< @'4 +'
<    44 4 $%(2)     4)%2)   '  4
 :-4 2+-
4 -; 44<'4 4-
5   @' 4    4 <     4  4 +
5+  < +4 <4 
  <<< '4'4 
-444 25:44'4'4 54
' + - 4 +5+4 25
20
;F -5+++'   +
 4  :<  ' 4444  +
4 5+)%2);4   
 :4'<   +5+$%(2) '
+<   5<4'- ;
'+ 5+$%(2)- -<
)%2)  4 <-''
4'F 44<    +5+ -
4):  <4'444 +5+$%(2
);<  5+ )%2)  '4 
4% ' 4 -
<4'+< +< 
5+4  5+ -4$%(2)6)%2) <  *

 + <5+ -+4$%(2)6)%2
) <  -    * 5  <  -  
44'4 44 45
<  4 :4--4'--
-4 -+4'4 
V24 ; 444 +B$%(2)6)%2)6
*C4 +B$%(2)6)%2)C<'4
21
E3'4:+5-< - 
-;4e(,,
22
E.'45++4)%$%( 
- 4-+ + 
++0+4 - -4e(,,
23
E1'45++4)%$%( 
 -*4-:+
< E3.;4e(,,
F +:E321;+  :F-3;4 
+  +5-+4< 
+)%2)+'$%(2)6)%2)
  $%(2) - -4   -5<  
-4 *
F  + ' 4     ++ :F- .;  
-4' +'+5+ )&<
 44<     '4 +-
5+)%2)6$%(2)<  *) 4<
 44-< 444)%2)$%(2)- -
4 )   - 4' +'-
'4'444 444)%2)F  -
24
4+44< '5 -+'
4+ '<    -<    4-
+5+-- 44 4 )%2)L *-
   5 <    + 5+  )%2) 6 $%(2) 
'4'444 '4'<
 +4  -5+<  * F 
-4- +'<-''4 444)%2
)
 +:F-.;-  '+4)+'$%(2) 
5 -4$%(2)6)%2)- 4
  +   <8 ++ < ++ 
-4-<+:<  -4< R
2; L   * -    44 4 - 5
5+<'4'
F   +  +   4 - <
 4 P[4  +++0
5+<++4 :E>;
5+  $%(2)6)%2):E7;  + 4 -R
+''4< +'-
 4   <      <
- 4 -' 5+ 
$%(2) $%(2)6 )%2)F  44-
5+ 5-<  -*:F-1
E,;
Table 7
Morphometric indices for the state of rat’s brain after repeated intraperitoneal
injections of NiO-NPs or Mn3O4-NPs nanoparticles, together or separately, and of
their combination with background oral administraon of the BPC (X±s.e.)
Neurons (%
%)
Control Mn3O4-NPs NiO-NPs NiO-NPs+
Mn3O4-NPs
NiO-NPs+
Mn3O4-NPs
and BPC
25
Nucleus caudatus
Without a
nucleolus
$,3,="11 .77,=17^6(1$.="(3^6.,$,="".^ $13=">76
With a
distinct
centrally
located
nucleolus
"3"=. 7$$=,7,^61,,=,(^6"$3=,73^ "$">=,76
Hippocampus (CA 1)
Without a
nucleolus
$,3,="$, 1,(3="$^ $3>=""61,(,=$13^ ($,="(^6
With a
distinct
centrally
located
nucleolus
(.(="7" $(=3^ $.=13^6,=$^ $,3=7.^6
Note:*. statistically significant difference from the control group; +. from the group given NiO-
NPs+Mn3O4-NPs (without the BPC); p < 0.05 (p < 0.05 by Student’s t-test with Bonferroni correction).
26
E>F -4 +++:*;&5'
4e(,,F +'+ < <2-
+ '- 
E7. F -45++4)%$%( 0
+++:*;&5'4e(,,4
<  +'0 
+-
27
E,. F - 4 5+ +4 )%$%(  
 -*  0 +++:*;&5'
4e(,,4:+<
E>7;
Table 8
The coefficient of genomic DNA fragmentation in the blood nucleated cells of rats
after repeated intraperitoneal injections of NiO-NPs or Mn3O4-NPs nanoparticles,
together or separately, and of their combination with background oral
administration of the BPC (X±s.e.)
Control Mn3O4-NPs NiO-NPs NiO-NPs+
Mn3O4-NPs
NiO-NPs+
Mn3O4-NPs and
BPC
BPC
,("=,,, ,3=,,^ ,3=,,^ ,3,=,,^ ,(3=,,^6,(=,,
Note:*. statistically significant difference from the control group; +.from the group given NiO-
NPs+Mn3O4-NPs (without the BPC); (p < 0.05 by Student’s t-test).
28
F 444  G) 4  -  :F->;
+-'4''M' 5+
 )- - *4' 
44 4   - 55+ <  *   +'
44 
4. Discussion
 < 4  5444 
 )    +      + 44' 4   -2
++5:*;&<   5'4 *
+  +' 4 ++ 4 <  4
   +  ' 4'   ' 4      44
-<   + 5+    5 +     +  
 44     +   +
G+ +4 <' 4 '+ 
  ' <'   4 - <      - 
++   <   ex juvantibus
: 4-4-4+
+4)<  -5+5'<F ++
<-'4+    *%2) 5' : 
",(;-  'M<E   4'  +  
 45445+in vitro -'
<   +454
E <++ 5+<  
4  - --' -42
5-+
F - ++44  
- 5+4)4- 5 
 +4'+5<   
4    <  )   5 <
'4'   -44
4   R -  - +  

29
L  + 444)%2)$%(2) < ' +
'4 '+4  <- +  
444$%(2)4 @'4 
        4   '   +   4
+ 5':F-3;<< + 4)%2
)<  '-<  +'45
:  +' +2-'4; 
F "( '545+)%2):F-$;<$
    .$"    +
4- 5+$%(2) 4
+4  -'' +4 R
 4)<5+ + 
+44-<4 + 5' '-
F 4- F-(+ +
44'4 * '     44 
+ <  +-'4'< 4
- 5  -*   4  
5+<  -+4 44' 4 
<'4'444 4'
 --4 
 ' -    4 < +< -
-4:4  
44'4'+<+;" +
4-':< 413?,,?4;-4 
++85+ -4+5+
 -< -*F M -
:<  -4'-<+;+
-,-< f*g f*-5+< *g'
(.-<f*g f*-5+<  4 
*g-M  ..-<  < 5++ < 
4'4  +5+ )
<  *- 4 +
2 AST, bilirubin, uric acid, thyrotropic hormone, SH-groups in the blood serum, monocyte and thrombocyte counts
30
44 4      4   + 5+    ) <  *

*+ 4444+- 5+)%2)
$%(2)  ''@-'4-
-'@-' % 5 +5
  4 44  - 5'   -  
: ",3-;-<   M 44
F 44 2+   4 '+4 - 5'4  )
- -2 -<<'
' <  -'< 
  < +'    ' 5+  $%(2) -   
-< )%2)<< -'
4 F 4+4 R
5'<<:",,3;5+5
4 <     4
+-'<  45 5:G77(;
M+-- <  44 4 -   
+ 45+A + 45
 <<  < 5+  4  <4 
<  4 +<
 < <5+:",; 
 5+ 4 -4 <
   4'M5+$%(2)
)%2)*+ 4<   -4
    < ' :     c2
4;  ++5+< 
  45+4 5'R 
5-'- 2  <4445+
-'-<  44  -
 4-  4<  5--' -
   + 5+    - 4 $%(2) )%2) <  
-5-2+
31
F - +4'4 45
)    4  :+ 4   ; 
+++F +  '+- <2<'
4 '- R   +4 
5 E < +4 
    4 - < 45+
$%(2)-< )%2):E>2,F-1;
+4'+'4 -
-     *4   +++ +<
-+4  + ' 4 ++ 4
   -  +' 4    <  < 4 +
< ++ '4   <-L    4'-
+ +'4++4 '+4R
544< +:2  
4+$;-<-  ++- 
E -+ '< ++4 
     - 5        ' 
++4  4'8:; 
5'4$%(2) 4 -4$%(2)
6)%2)0 :-;  < 4 ' 5' 4 )%2)  +'0 :;
4 444-5'-'-*

  -+44< -4+44
:  + 4 ;<  <' 4'
 '  5+$%(2)-  -< )%2
)L - 44- 5+5)
:  ",;  : ",$;
++5:",(;
F 4+ '-' +4+
-' 4 -@4 #/++'
)%2)4 <  +<  4$%(2)< '
32
 '4 +44<
< <+ +'+ +4++4
R44+++-::4
",,";; @ +44 +' +:*-'
",,$;F +44      4 
)R +'544<  <-< ++:
<-'  ",$;F 4 44 4 )-'-
4-2+ 5':-'5+
< 2)*%2)<;+44 +L
  M4' <  +'R     +4  
 ) - ' 4 ++ -4  -  
-' '4 ++4':F-
.; 54)24 +'<    
5+)%2)< <@ + <-'
 +44F +'4 <44
 - 44+     4
+'-'$%(2)-')%2)< 44
4<  -444 ) +44
5+'+<  ' +' -'<'
- + 44   <  <  ' 2
4< -*:F-.;
F 444 * + 4-
+< 4 -< 4 5
:;     4 4 )%2) :F- " E $  (; F  
544+-  444 *4 
+ 4 +R+ 4 :F-
.;L-  ++)25+4 4
  +R  ++ 4 <   <   +-' 
+ 4-'4)-4+'
' '  <  <   + 4    F  '+  ' -
--' 4  *  '-'
- 5 5+     +R  <  <  <
:F-(;
33
   -  <   <  - 4<  5+' -4
toxicokinetic 44 4 *<  4     4
<     4 toxicodynamic 44 4 4   *
-+'< ' + -5
5+    4   *    :   ;  
4 R--< +''4' 
 <   4< 4 F- 1   * -' 
+       -  4     -
5+<   5+<+--'<  
5'4 <  4 -5
 <- - 5+
*%2) :",(;      5+< -
4  44 4 4 * <+-'
 ++4 -< 
L-   -<  +44'
4 *   4445  <:  +5;
+4 45  *+
 4 -45'444-+< 
  :+'  +- " <  @4'    4
-+;<    44
4'- '
<4M5 
4'  44 544 R
:<-'",(; +45+ *
    -' -       4   44 
-4  54 < <   -
 4 '
 -'-' 454
: ; 5+ 5+)%2) 
 +54< 5+$%(2) 
- 54
5<4'<5+ -4  
)%2)< 5 '<4'   
34
5+$%(2)F - '+ '
5+  4< F5     '  @  -
5++-' -'- &<
 54  + 
  < +' ' '2-+4   -'  
- '   4 ' '    44 4 
: -' F     7>7;      5
-+4      + - 
'*4 '4  '+ 
 - '25444-5+
)%2) 6 $%(2) <  -'   * <    -  
 ':F-3;  ' ':F-(;
#< -4M'+4'
-<55'4 444-+
4<    44 <2-  -4
%(6)*":",3; +44
-4+- 54
 -'-'5 4+
)4-')R -4  
-444 ) 4 44-' F  +5  
'-- 4  
+'+4 44-- 
:F-";  4 44)%2
)< <-444 
 +:E(;
F ' <44'4$%(2)
 < 4  
 < <       - 5+ -' 4
in vivo 45++< 5.
F  '+  < ++     in vitro 5+ -'
+ 4 -' 4$%(2))%2)-
-4"(  4$%(2)3
<   4)%2)  -'1?'F  '4#/
  < 48 4 $%(2) -' <25+ 4 < 
35
τ1h",=$ τ2h("3=,,< 4)%2)-'5+
4< τ h(,3=1,
5'4'+<-'4 
  'in vitro 5+-+<
<-<   +4 /G4in vivo
  G) 4 R  and 4  -  
- 5+  ) ) : ",$; ++
5):",(; 5+< 2)*%2)<
+  G)2 44 5+   )
 * +  +': 
44  ; L4  -   44 < 4'
F 5++<- /G2 
 '<+4'-:F->;BC4 in vivo
5' 4)+'
-4-' *
'<'4 4< 
4   ' 4  4  + <       <
-4.4 +*'8
 4-G&''+ ' 
4FF '5-F + '
+4  4 '4'- -
  -- '++-  '
F   *   ' 54   - 25+ 
5+4 4/*0< 4
 -'- @
&<+ +4 + *
+ - 5 5+ <   +5+   -
< * <  444'
44 + 4 4 *  '5
4 -< 4  -24  
++-  -5+'-4
36
F  5+"< @4' +4
 -+'  4  -'
 -4 -54 
-M@4 45-' 
5+    +   - 4    
  4   *   @     4  5+
:  ",(;<-   5+  +
44'-44'2444 *
5. Conclusions
%5+  < - 5+4+ +
4 :;4:;5 4 + 4
' 5+- 5 <   -  -'
 -4' 44
' '+ + 4 &<
 @'4 $%(2)5 )%2)+4 
4 4:- +' 44  
);  /#:+;<-'<+
 +4$%(2)  4+ +4
)%2)i+--'-    5' <4 $%(2
)    <    -'+< )%2
)
 5+$%(2) 4
 -  444 4   
+++-'    -' 4)%2)
-5'+4
F '+4-5'4$%(2))%2)44444
:' '  -';   4  +
 '% <  < -  -
5+++45
 - 5+$%(2)
37
-  4   44     - 5+ -'  
4  4 -+   4 <     44
'+    4 5'  + ++ ' ++ - 
+ '4-   ' 4   44 
    + 4   
Acknowledgements
F M+4 D*4 DB 'C4 D
ED'<  <  <+'++-' '
4#4 /E:DG/E#E37((Y,,;
Conflict of Interest
F  44
References
@2G 2 EE'  *
22G+ '%S2*GS",(/45
+ +  - 4<E-
F5"$
-EM",(
-   5' 4 4    5
++-'  +S)777Q
7"7
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4544+ F5"".:;">2$(
*-'E#& & L-)-   E
S",,$+4444++- 
'5+&+'$>>>Q7>
G#+ /G/E
)#771 4'+5'
-*" /$.:(;">32"7"
G#+ /EG#
77(G+' '4    
38
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5* G:$";((32(3
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4 (7* )L
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1L&%
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!% ",-2  '5' -24 E$%(
2+4<++S
F5$,.,Q.1
    G' FG   -' S
",,> B ++ '5CU% 4   <'  + 4   '
+'  ++  +*#S %+
#((Q("
  ' %&   )   G' FG
      %! '  F ' 
",,1 F 4-+  5 44 4 -4
5*#S%+#$"3Q".(
39
 !%'%&G'FG
 %!  #     ' #
 G##')
 ",,7 F5' 4+< +54-2
+,,(33Q(1,
    G' FG   ",,
#5+4  5'4  5E$%( :;+
*#S%+#.(1Q.$
  
 ",( < <' +      -  
 4    +    < -2
++2F.>2(:/;
         ' #
",(# +R/F5#5+
5'F4 G #%+ & /:42
%<;S#3($32((7
 '%& !S
'#)",$*+
   4   -244 4 M   
+  4R44< +54
-+S("((7Q"(>$
          )
'#,   !  #!'
%&#",344in vivo 5+< 
5+8 +'+ '+- '
5'  5' ' ++   4 -+ : 42
<;S),$,$2$,"7
')
' F-",3-E +4  '
40
 +4-5'8++ 4''+4
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42
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