Effect of membrane lipid peroxidation on rat liver microsomal enzyme activity

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(1)³¤¥Èó. * F* a. (2004) 44 2 Korea J Vet Res(2004) 44(2) : 185~193. ïæî"Özf*^ÚîÂ5Ò{æBVË"~ ç&Wö&. ;ç*Á«ê. *§&v >~"& Ún;W² (²Òß: 2004j 4ú 22¢) Effect of membrane lipid peroxidation on rat liver microsomal enzyme activity Sang-Youel Park* and Jong-Hoo Cho Bio-Safty Research Institute, College of Veterinary Medicine, Chonbuk National University, Jeonju 561-756, Korea (Accepteda April 22, 2004) Abstract : The effects of membrane lipid peroxidation and retinyl palmitate on rat liver microsomal functions were investigated in vitro. Rat liver homogenates exposed to oxygen tension for 0, 3, 6, 9 or12 hours and lipid peroxidation levels were evaluated by the measurements of fluorescence intensity, malondialdehyde (MDA) and retinyl palmitate. The fluorescence intensity of homogenates and microsomes were elevated and retinyl palmitate concentrations were decreased. But the concentration of MDA was not affected to exposure time. Therefore, fluorescence intensity and retinyl palmitate concentration were used to analyze the correlation between lipid peroxidation and microsomal functions. To investigate the liver microsomal functions, the microsome was isolated from rat liver homogenates exposed to oxygen. The concentration of cytochrome P450 and the activity of NADPH-cytochrome P450 reductase in liver microsomes were gradually decreased with increasing the exposure time. The correlation between fluorescence intensity of microsomes showed a very high inverse correlation of -0.97 and -0.93, respectively. The decrease of cytochrome P450 concentration was due to the regeneration of cytochrome P450 to cytochrome P420. Also, the activities of cytochrome P450-dependent aminopyrine demethylase and benzpyrene hydroxylase of liver microsomes were gradually decreased with increasing the exposure time. The correlation with fluorescence intensity of microsome showed a high inverse correlation of -0.97 and -0.91, respectively. The retinyl palmitate concentrations of rat liver homogenates were decreased with increasing the exposure time. The decrease of retinyl palmitate concentration was followed by a low concentration of cytochrome P450 and activity of NADPH-cytochrome P450 reductase. The correlation indicated high direct correlation of 0.92 and 0.93, respectively. The decrease of retinyl palmitate concentration was also accompanied by the reduction of aminopyrine demethylase and benzpyrene hydroxylase activities. The correlation was analyzed a high direct correlation of 0.90 and 0.85, respectively. In conclusion, these studies have shown that the membrane lipid peroxidation of rat liver microsome proportionally decreased microsomal enzyme activities in vitro experiments. Key words : microsome, retinyl palmitate, malondialdehyde, cytochrome P450. ¢^f 2003jê *§&v Ún;W²~ ¢¦ æöb >îr *Corresponding author: Sang-Youel Park College of Veterinary Medicine, Chonbuk National University, Jeonju 561-756, Korea [Tel: 82-63-270-3886, Fax: 82-63-270-3780, E-mail: [email protected]]. 185.

(2) ;çÁ«ê. 186. B . ÿbÚ~ ^f ^ ²V& ~ ïf " ®z æOÖb WB æî 7[ Òö Wî Ú Ò~¾ 6º ïö Â>Ú / Wî, OÚ Wî 6º N >ÇêB~ j ~, ®z æO Öf >wW Ö²«(reactive oxygen species, ROS)ö ~ ~ £² Öz>Ú æî "Özbj W~ "æ ç~ ¶ç" æWj .¾~ ^ VËj ¶çÎ. [2, 6]. z~ "º º7ö ~¾ >wW Ö²«ö ~ WB æî "Özbf »Z>Ú malondialdehyde (MDA) 6º 4-hydroxynonenal" ?f aldehyde~¢ W~ [3, 6], 2N'b ^Ú jV¢ &æ ® º æî, Wî 6º Ö" ?f bî" Ö~ &ª¶¢ W~, ß® Ö" Ö rº òæ ¢ FB~ zj ¢bÒ > ® >î [4, 13]. ^Ú~ ²Úº £®4³£" ?f Fbî 5 æî j >ÏW bî *~Êº ë Î²ê mixed function oxygenases (MFO) Êj &æ ® b æî Wö &~º 7º ²V&b, ^ ¢ 2ê~ ªÒ ²Ú~ «¶¢ îÂ (microsome) ¢ . îÂ~ MFO Êf NADPH ¦V *¶¢ *~ cytochrome P450j ~ öÚb ÖB æÏW FVzbö Ö²¢ B~ hydroxylV¢ <º >ÏW bî *~B Ú ~ VJj Ï~² [7, 12]. *¶* " Ö²~ ÒÏ";öB ROS îÂöB W >Ú ïæî "Öz¢ FBÊº Ò þ&Ú. þöB {>îb [9, 11, 17], Ú þöBê ïæ î "Özö ~ îÂ VË æzö & & ® [1, 8, 16]. ÚöB ROSº ê³~ W>, ROSö ~ ïæî "Öz& ê¯>V r^ö Ú~ ;Fæ¢ *~ ïæî "Öz& ²V& ~ FVËö ~º 'Ëj «~º ©f Ö 7º~. 'B . V¢B öBº Ñ *^ îj ¢; Ö ²{~öB ïæî "Öz¢ FêÎ ê îÂj ªÒ~ ïæî "Öz >&j MDA G;»" ;7ª C»b ï&~&b, ö >>>º îÂ VË ~ ï&¢ *~ MFO Ê~ bî cytochrome P450 ·" cytochrome P450 reductase Wj ï&~&. . jÞ B zb~ &ÒËKj ï&~V * ~ cytochrome P450 ~ aminopyrine demethylase W" benzpyrene hydroxylse Wj G;~&b, ï æî "Özö ~ ;7bî~ W" retinyl palmitate ³ê æzf~ &êê «~¶ ~& .. Òò 5 O». þÿb F çê~ Sprague-Dawleyê > Ñ 30îÒ¢ *§&v Ún;W²öB ª·Aj â·Òò (")öB B îÖÊ Òò¢ b" þ Bì / ~B þÿb öB ÒG~& . £ 4Bú ÒG ê Ú7 £ 250 g *êö ê~&j r v¦¢ æÏ~ ~ÒÎ ê *j j~ þö ÒÏ . ªC£ 5 ªCVV îÂ~ cytochrome P450" NADPH-cytochrome c reductase W G;j * 1,2,4-aminonaphtol sulfonic acid, cytochrome c 5 NADPHf îÂ~ £b &ÒG;j * aminopyrine" benzpyrenef Sigma (U.S.A.) B®j ÒÏ~&, malondialdehyde G;j * thiobarbituric acid (TBA)f trichloroacetic acid (TCA) º Lancaster (England)B®, Væ ¢> £f Junsei (Japan) B®~ 1/ £j ÒÏ~& . ªCVV îîêVº SMT process homogenizer (SMT Co., Japan)f teflonpestle homogenizer (Wheaton, U.S.A.)¢ ÒÏ~&, îÂ~ ªÒ¢ *Bº super speed centrifuge (Vision, Korea)¢ ÒÏ~&b, 7ê G;f UV-2100 spectrophotometer (Shimadzu, Japan)¢ ÒÏ~&, ;7 Ê¿Þ"" ;7;ê~ G; f spectrofluorometer (SFM25, Italy)¢ ÒÏ~& . *^ îÂ~ ªÒ Ñ¢ ~ÒÎ ê ¯ *Ëj B~ ^.~ ïË Tris jÏ (0.25 M sucrose, 20 mM nicotinamide, 27 mM KCl, pH 7.4)b 3² ç ^¿~ ÖÚç " wB .j B~& . ^¿ çö ?f Tris jÏ 30 ml¢ &~ SMT process homogenizer 1ª* çj 2ê Î ê teflon-pestle homogenizer¢ ÒÏ~ 0-4 C¢ Fæ~B 4² stroke ¢ ~ î ¦Fj þö ÒÏ~& . î Â~ ªÒº î ¦Fb¦V Æ~Òj¢ ª Ò~ B~ Omuraf Sato [15]~ O»ö &~ î Âj ªÒ~& . * ";j º£~ î ¦F j 460ÜgöB 5ª* öªÒ~ ^ 2êb" *bj ªÒ~ B~ çÓj 4,300Ügö B 10ª* öªÒ~ ^ 2êb *j B~ & . áÚê çÓj 12,500ÜgöB 30ª* öªÒ ~ Æ~Òj *bj ªÒ~ B ê áÚ ê çÓj 100,500ÜgöB 90ª* öªÒ~ î Â *bj ªÒ~& . îÂ *bj Tris o.

(3) ïæî "Özf *^Ú îÂ 5 Ò{æB VË"~ ç&Wö & . jÏö ¦FB ® teflon-pestle homogenizer ¢ ÒÏ~ îÂ î ¦Fj áî . *f ? áÚê * î" îÂ ¦Ff Wî ³ ê¢ G;~ jºö V¢ ¯ '. ³ê C~ þö ÒÏ~&b, ¦ & jº& ®j r º ïÿ &~ 3¢ Úö þö ÒÏ~& . ïæî "Öz >&~ G; (1) Malondialdehyde (MDA)~ G; Keller [10]~ O»ö &~ r" ? G;~&. . TBA £ (15% TCA, 0.375% TBA, 0.25 N HCl) 2 mlf î 6º îÂ ¦F (1 mg protein/ml) 1 ml, Ò 10 µl~ 10% BHT¢ &~ b~ ¶ j >öB 15ª* & ê ï'Î >wj 3,000 ÜgöB 10ª* öªÒ~& . áÚê ç[~ ;7 ;ê¢ V2Ë (Ex., Excitation wavelength) 515 nmf OÒ2Ë (Em., Emission wavelength) 553 nmöB G; ~& . &®b tetramethoxy propanej çVf ?f O»b ¾Ò~ áf MDA & ;7Fb¦V MDA ·j ÖÂ~& . (2) ;7;ê~ G; * î" îÂ ¦Fj Tris jÏb C~(2 mg protein/ml) Ex. 342 nmf Em. 455 nmö B ç&' ;7;ê¢ G;~ jv~& . 3FUJOZM QBMNJUBUF G; î7~ retinyl palmitate ³êº Szweda [18]~ O »ö &~ G;~& . ¯, * î C (6 mg protein/ml Ï 50 µl)ö methanol 1 ml¢ &~ ¾ b ê 1000ÜgöB 5ª* ö ªÒ~& . çÓ 1 mlö methanol 2 ml¢ &~ b, 3V CB Ex. 330 nmf Em. 471 nmöB ç&' ;7;ê¢ G;~ & retinyl palmitate¢ ¾Ò~ ·W & ;7 Fb¦V î 1 ml (1 mg protein F) retinyl palmitate ·j ÖÂ~& . îÂ W~ G; (1) Cytochrome P450 G; îÂ ¦F 7 cytochrome P450 ·f Omura f Sato [15]~ O»b G;~& . ¯, îÂ ¦ F (2 mg protein/ml, 0.15 M KPO jÏ) 5 mlö sodium dithionite¢ ²ï &~ ¾ b~ 2.5 ml O v B~ jï&ö ¾*Ú I ã jï&ö ¢Özê ²¢ zB -æ pf jï&j &~ 2Ë 380 nmf 500 nm Ò~ 7 Ê¿Þ"j ·W~& . 4. 187. f öB~ 7ê N¦V j Ï~ ~ ³ê¢ ÖÂ~&. (2) NADPH-cytochrome c reductase W G; îÂ ¦F NADPH-cytochrome c reductase Wf cytochrome c Ï (5 mg cytochrome c/ml, 0.3 M ÖjÏ, pH 7.5) 0.2 mlö 0.3 M ÖjÏ 0.8 ml, 9.5 mM NADPH 10 µl, îÂ ¦F (5 mg protein /ml) 10 µl¢ 1 ml jï&öB b~ 1ª ê 2Ë 550 nm öB 1ª* 7ê æz¢ G;~& . 1ª*~ 7ê æz¦V ~ö cytochrome c~ extinction coefficient, 21Ü10 µmol min mg proteinj Ï~ ~ö> º cytochrome c~ nm/min/mg proteinb Wj ~& . (3) Aminopyrine N-demethylase W G; Wills [20]~ O»ö &~ G;~& . ¯, 0.05 M Tris-HCl jÏ (pH 7.5) 3 ml, îÂ ¦F (5 mg protein/ml) 1 ml, 9.5 mM NADPH 20 µl, Ò 0.1 M aminopyrine 1 ml¢ b~ 37 C~ >öB >w ÊB 5ª *Ïb 4²ö ö >w 1 ml O ~ 10% TCA 1 mlf b~& . bj NöB 15ª* O~ ê, Nash £ [14] 2 mlf b~ 60 C N>öB 15ª* >wÎ ê 2,500 r.p.m.öB 15 ª* öªÒ~ ç[~ 7ê¢ 2Ë 412 nmö B G;~& . Î²ö ~~ WB formaldehyde · j &Fb¦V ÖÂ~ Wj W formaldehyde µmol/min/mg proteinb ~& . (4) Benzpyrene hydroxylase W G; Watanabe [19]~ O»ö &~ G;~& . º£ ~, îÂ ¦F (2,5 mg protein/ml) 0.2 ml, 0.05 M Tris-HCl jÏ (pH 7.5) 0.8 ml, 9.5 mM NADPH 10 µl, Ò 0.01 M benzpyrene-acetone Ï 10 µl¢ ¾ b ê, 37 CöB 15ª* >wV . >wj ;æ ÊV *~ acetone 1 ml¢ Î&~, hexane 3 ml ¢ Î&~ 10ª* ºÂ~& . Hexane 1 ml¢ ~ 1 N NaOH 3 mlf b~ 1ª* êû~ 4,000 r.p.m. öB 5ª* öªÒ~& . NaOH [j ªÒ~ Ex. 396 nm, Em. 522 nmöB ÖzB benzpyrene~ ;7; ê¢ G;~& . Wêº ÖzB benzpyrene~ nmol/ min/mg proteinb ~& . Ûê'ªC þöB áÚê þ &G~º 5² > þ~ 450 nm 490 nm cytochrome P450 extinction coefficient, 91 mM−1cm−1 (nmol cytochrome P450/mg protein).. −1. 3. −1. o. o. o.

(4) ;çÁ«ê. 188. ïÛ&ÞN ¾æÚî . F~W ¦;f Student t¦;ö ~~&b, ç&ªC»ö ~~ ²æO;" ç&ê>(r)¢ ~& .. Ö ". * î~ Ö²¾Ò& * î 5 îÂ ~ ïæî "Öz>&" SFUJOZM QBMNJUBUF ³êö ~º 'Ë (1) ;7;ê~ æz * î 5 îÂ ¦FöB ;7Ê¿Þ"~ N& ®ºæ¢ {~V *~, '' 2 mg protein/ ml C~ ·W Ex. 342 nmöB~ ;7Ê¿Þ" f Fig. 1" ? Îv Em. 455 nmöB ;7 peak¢ ¾. æÚî . V¢B æî "Özö V ;7;ê~ æz º Ex. 342 nmf Em. 455 nmöB G;~ jv~& . * îj Ö²{~ö 30 C >öB êû~B 3 * *Ïb 12*ræ 5²ö ö j~ î Âj ªÒ ê Wî 2 mg/mlj F~ê C * î" îÂ ¦F~ ç&' ;7;ê ¢ G; :, Fig. 2f ?~ . * î" îÂ ¦F ÎvöB 12*ræ ;7 ;ê& Ã&~&rj r > ®î . (2) MDA ï æz Ö²¢ ¾Ò * î" îöB B î Â ¦F~ MDA ï~ æzº Fig. 3" ?~. . * î~ MDA ïf 3* Úö Ã&~&b o. Fig. 1. Fluorescence spectra of homogenates and microsomes from rat liver homogenates exposed to oxygen tension. Emission spectra excite at 342 nm were recorded.. Fig. 3. Malondialdehyde concentration of homogenates and microsomes from rat liver homogenates exposed to oxygen tension. Each value is the mean ±SD for 5 repeated experiments. *P<0.05 vs. 0 hour.. Fig. 2. Relative fluorescence of homogenates and microsomes from rat liver homogenates exposed to oxygen tension. Each value is the mean ±SD for 5 repeated experiments. *P<0.05 vs. 0 hour, **P<0.01 vs. 0 hour.. Fig. 4. Effect of incubation time on hepatic retinyl palmitate concentraion in homogenates exposed to oxygen tension. Each value is the mean±SD for repeated experiments. *P<0.05 vs. 0 hour..

(5) ïæî "Özf *^Ú îÂ 5 Ò{æB VË"~ ç&Wö & . Fig. 5. The spectrum changes of microsomal cytochrome P450 from rat liver homogenates exposed to oxygen tension.. ê æì ÿ¢ >&j Fæ~& . î Â ¦FöBº 12* êö ² çß ©j B~ º F~W ®º æz¢ æ p~ . (3) Retinyl palmitate ³ê æz ÚöB ïæî "Öz& ê¯>B retinyl palmitate & Ã&~& º ö V¢ * îj Ö² ¾Ò ~ retinyl palmitate ³ê~ æz¢ Ò : Fig. 4f ?~ . Ö² ¾Ò *ö V¢ îöB Îv retinyl palmitate~ ³ê& 6²~V ·~ ¾Ò* ^Ú öö V¢ ê³ 6²~& . Ö²ö ~ ïæî "Öz>&" SFUJOZM QBMNJUBUF ³ê æz& îÂ VËö ~º 'Ë (1) îÂ ;7;ê æz& MFO Êö ~ º 'Ë îÂ~ ïæî "Özö V ;7;ê~ Ã& & MFO Ê~ bî cytochrome P450 ¶Úö ~º 'Ëj Ò~¶ cytochrome P450~ Ê¿Þ" j ·W~ Fig. 5f ?f Ö"¢ áî . Ö² ¾Òº 2Ë 450 nm peak¢ ¾æÚº cytochrome P450~ 6² f þ 2Ë 420 nm peak¢ ¾æÚº cytochrome P420 b~ *~j "îb, º cytochrome P450 Ö²ö ~~ 2Z>æ p cytochrome P420b æ W>º ©j ~ . Ö²¾Ò*ö V îÂ ¦F cytochrome P450~ ·" cytochrome P450ö *¶¢ *~º NADPH -cytochrome P450 reductase W~ æzº Fig. 6Af ?. . Cytochrome P450 ·" NADPH-cytochrome P450 reductase W Îv Ö²¾Ò * ã"ö V¢ 12. 189. * ÿn ê³ ÿ> 6²~& . îÂ ¦F~ ;7;ê Ã&f cytochrome P450 · 5 NADPHcytochrome P450 reductase W"~ ç&Wj Ò : Fig. 6B 5 Fig. 6Cf ?~ . îÂ ¦F~ ; 7;ê& Ã&ö V¢ cytochrome P450 ·" NADPHcytochrome P450 reductase W Îv 6²~ cytochrome P450 ·"~ ç&ê>º r =-0.97, NADPH-cytochrome P450 reductase W"~ ç&ê>º r=-0.93~ Ö ¸ f ç&Wj ¾æÚî . * îö Ö²¢ ¾Ò Ö" retinyl palmitate ³ ê& 6²~ MFO Ê 7 îÂ~ cytochrome P450 ·" NADPH-cytochrome P450 reductase W 6 ²>îbæ B ç&W FZ¢ Ò~ Fig. 6Df Fig. 6E~ Ö"¢ áî . ¯, retinyl palmitate ³ê 6²º cytochrome P450 ·~ ÿ> 6² ãËj &b, r=0.92~ ¸f ;ç&Wj & . NADPHcytochrome P450 reductase Wê r=0.93b retinyl palmitate ³ê 6²f þ ² &~>º ;ç&W j ¾æÚî . (2) îÂ ;7;ê æz& aminopyrine Ndemethylase 5 benzpyrene hydroxylase Wö ~º 'Ë * îö Ö²¢ ¾Ò~ 3* *Ïb 12* ræ 5²ö ö G; îÂ~ cytochrome P450 ~ aminopyrine N-demethylase W" cytochrome P450 ~ benzpyrene hydroxylase W~ æzº Fig. 7Af ? . v Î² Îv Ö²¾Ò * ^Úöö V ¢ W &~~&b benzpyrene hydroxylase~ W z Â] &~¢ & . * îö Ö²¢ ¾Ò~ îÂ~ ;7;ê& Ã&~&bæ ; 7;ê~ Ã&f cytochrome P450-~ Î² W 6² *~ ç&Wj Ò : Fig. 7B 5 Fig. 7Cf ?~. . Aminopyrine N-demethylase W 5 benzpyrene hydroxylase Wf îÂ~ ;7;ê~ Ã&ö > ~ 6²~&b '' r=-0.97" r=-0.91~ ¸f ç &j ¾æÚî . * î~ Ö²¾Òö ~ * î~ retinyl palmitate ³ê 6²f îÂ~ aminopyrine Ndemethylase W 5 benzpyrene hydroxylase W 6² *~ ç&Wj Ò : Fig. 7D 5 Fig. 7E f ?~. . Ö²ö ~ * î 7 retinyl palmitate ³ê~ 6 ²º îÂ ¦F~ aminopyrine N-demethylase W" benzpyrene hydroxylase Wj &~B ' ' r=0.90 5 r=0.85b Ö ¸f ;ç&Wj ¾æ Úî ..

(6) ;çÁ«ê. 190. Fig. 6. A, Cytochrome P450 concentration and NADPH-cytochrome c reductase activity of microsomes from rat liver homogenates exposed to oxygen tension. Relationship between B, relative fluorescence and cytochrome P450 concentration, C, relative fluorescence and NADPH-cytochrome c reductase activity, D, retinyl palmitate concentration and cytochrome P450 concentration, E, retinyl palmitate concentration and NADPH cytochrome c reductase activity of microsomal suspension from rat liver homogenates exposed to oxygen tension. Each value is the mean±SD for 5 repeated experiments. *P<0.05 vs. 0 hour, **P<0.01 vs. 0 hour.. V. * î~ Ö²¾Òº îÂ ¦F~ ;7;. ê¢ Ã&Vb¾, î 7 retinyl palmitate ³ê¢ 6²B æî "Özö V ;7Wbî~ W" Ò ?f ;7W bî retinyl palmitateº î'b.

(7) ïæî "Özf *^Ú îÂ 5 Ò{æB VË"~ ç&Wö & . 191. Fig. 7. A, Aminopyrine N-demethylase activity and benzpyrene hydroxylase activity of microsomes prepared from rat liver homogenates of oxygen incubations. Relationship between B, relative fluorescence and aminopyrine N-demethylase activity, C, relative fluorescence and benzpyrene hydroxylase activity, D, retinyl palmitate concentration and aminopyrine Ndemethylase activity, E, retinyl palmitate concentration and benzpyrene hydroxylase activity of microsomal suspension from rat liver homogenates exposed to oxygen tension. Each value is the mean±SD for 5 repeated experiments. *P<0.05 vs. 0 hour, **P<0.01 vs. 0 hour.. 2Z>º ©b {>î . öBº þ& Ú Ö²¾Òö ~ * î~ ;7;êf retinyl. ³ê Ò îÂ VË Ò~ ç&& ê¢ rj¶ ~& .. palmitate.

(8) ;çÁ«ê. 192. îÂ~ MFO Êf ëV*j >¯~, >º *¶>ÏÚº cytochrome P450 *¶> Ïj / ~º Î²º NADPH-cytochrome P450 reductase . V¢B öB Ö²¾Òö ~ ;7;ê~ æzf cytochrome P450 · 5 NADPH-cytochrome P450 reductase W*~ ç&Wj Ò Ö" cytochrome P450 ·" NADPH-cytochrome P450 reductase W Î v ;7;ê~ Ã&ö >>~ 6²~º ç&~ & ê¢ ¾æÚî . Ö"º Gut [5]~ öB ïæî "Öz~ Fêº cytochrome P450 ·j 6² V º Ö"f ¢~~º ©î . NADPH-cytochrome P450 reductase~ Wê cytochrome P450~ æWö ~ ~ 6º Î²¶Ú~ æWö V¢ &~>º ©b 'B . Cytochrome P450 ·~ 6²º æWö ~~ cytochrome P420b *~>º ©b {>îb Karuzina f Archakov [9]~ öB "Öz>²ö ~ ïæî "Öz Fêº cytochrome P450j 2Z~ 6²>î º ÚÏ"º ©î . jÞ ç7 £ bj &Ò~º cytochrome P450-~ aminopyrine Ndemethylasef benzpyrene hydroxylase~ Wê Ö²¾ Òö ~ æî "Öz >& Ã&ö V¢ &~>Ú ç&~ &ê¢ ¾æÚî . Ö"º þ' ï æî "ÖzFêº £b&Ò¢ ÛB V º Wills [20]~ f ¾ ¢~~, ïæî "Öz& î Â~ VËö 'Ëj ~º ©b 'B . Ö'b þ&Ú þöB * î~ Ö²¾Ò ö ~ retinyl palmitate ³ê~ 6²º MFO Ê ~ cytochrome P450 ·" NADPH-cytochrome P450 reductase~ W6²¢ >>~& . 6 cytochrome P450-~ aminopyrine N-demethylasef benzpyrene hydroxylase~ W 6²>º ãËj ¾æÚî . ¾ retinyl palmitate ³ê 6²ö V îÂ VË~ & ç7 retinyl palmitate ³ê ¶Úö V ~º ©æö &~º {B & ìb, ö & ê³ & jº ©b 'B .. ^^ò. 1. Ando, M., Katagiri, K., Yamamoto, S., Wakamatsu, K., Kawahara, I., Asanuma, S., Usuda, M. and Sasaki, K. Age-related effects of heat stress on protective enzymes for peroxides and microsomal monooxygenase in rat liver. Environ. Health Perspect. 1997, 105, 726-733. 2. Ashok, B. T. and Ali, R. The aging paradox: free radical theory of aging. Exp. Gerontol. 1999, 34, 293-. 303. 3. Burcham, P. C. and Kuhan, Y. T. Introduction of carbonyl groups into proteins by the lipid peroxidation product, malondialdehyde. Biochem. Biophys. Res. Commun. 1996, 220, 996-1001. 4. Filser, N., Margue, C. and Richter, C. Quantification of wild-type mitochondrial DNA and its 4.8-kb deletion in rat organs. Biochem. Biophys. Res. Commun. 1997, 233, 102-107. 5. Gut, J., Kawato, S., Cherry, R. J., Winterhalter, K. H. and Richter, C. Lipid peroxidation decreases the rotational mobility of cytochrome P-450 in rat liver microsomes. Biochim. Biophys. Acta. 1985, 817, 217228. 6. Halliwell, B. and Chirico, S. Lipid peroxidation: its mechanism, measurement, and significance. Am. J. Clin. Nutr. 1993, 57, 715S-724S; discussion 724S725S. 7. Handler, J. A. and Brian, W. R. Effect of aging on mixed- function oxidation and conjugation by isolated perfused rat livers. Biochem Pharmacol. 1997, 54, 159164. 8. Hayashi, T. and Miyazawa, T. Age-associated oxidative damage in microsomal and plasma membrane lipids of rat hepatocytes. Mech. Ageing Dev. 1998, 100, 231-242. 9. Karuzina, II and Archakov, A. I. Hydrogen peroxidemediated inactivation of microsomal cytochrome P450 during monooxygenase reactions. Free Radic. Biol Med. 1994, 17, 557-567. 10. Keller, J. N., Kindy, M. S., Holtsberg, F. W., St Clair, D. K., Yen, H. C., Germeyer, A., Steiner, S. M., Bruce-Keller, A. J., Hutchins, J. B. and Mattson, M. P. Mitochondrial manganese superoxide dismutase prevents neural apoptosis and reduces ischemic brain injury: suppression of peroxynitrite production, lipid peroxidation, and mitochondrial dysfunction. J. Neurosci. 1998, 18, 687-697. 11. Lenaz, G. Role of mitochondria in oxidative stress and ageing. Biochim. Biophys. Acta. 1998, 1366, 53-67. 12. Longmire, A. W., Swift, L. L., Roberts, L. J., Awad, J. A., Burk, R. F. and Morrow, J. D. Effect of oxygen tension on the generation of F2-isoprostanes and malondialdehyde in peroxidizing rat liver microsomes. Biochem. Pharmacol. 1994, 47, 1173-1177. 13. Marnett, L. J. Lipid peroxidation-DNA damage by malondialdehyde. Mutat. Res. 1999, 424, 83-95..

(9) ïæî "Özf *^Ú îÂ 5 Ò{æB VË"~ ç&Wö & 14. Nash, T. The colorimetric estimation of formaldehyde by means of the Hantzsch reaction. Biochem. J. 1953, 55, 416-421. 15. Omura, T. and Sato, R. The Carbon MonoxideBinding Pigment of Liver Microsomes. I. Evidence for Its Hemoprotein Nature. J. Biol. Chem. 1964, 239, 2370-2378. 16. Pierrefiche, G. and Laborit, H. Oxygen free radicals, melatonin, and aging. Exp. Gerontol. 1995, 30, 213227. 17. Plewka, A., Kaminski, M. and Plewka, D. Ontogenesis of hepatocyte respiration processes in relation to rat liver cytochrome P450-dependent. 193. monooxygenase system. Mech. Ageing Dev. 1998, 105, 197-207. 18. Szweda, L. I. Age-related increase in liver retinyl palmitate. Relationship to lipofuscin. J. Biol. Chem. 1994, 269, 8712-8715. 19. Watanabe, M., Tanaka, M., Tateishi, T., Nakura, H., Kumai, T. and Kobayashi, S. Effects of the estrous cycle and the gender differences on hepatic drugmetabolising enzyme activities. Pharmacol. Res. 1997, 35, 477-480. 20. Wills, E. D. Lipid peroxide formation in microsomes. Relationship of hydroxylation to lipid peroxide formation. Biochem. J. 1969, 113, 333-341..

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