Olive oil polyphenols modify liver polar fatty acid composition and inhibit CCl4-induced hepatotoxicity in Balb/c mice

Olive oil polyph e n ols m odify live r pola r fa t t y a cid com posit ion a n d in h ibit CCl 4 - in du ce d h e pa t ot ox icit y in Ba lb/ c m ice indu Jasminka Giacomettia, Hrvoje Križana, Neven Franjićc and Alena Buretić-Tomljanovićb Universit y of Rij eka, School of Medicine, aDept . of Chem ist ry and Biochem ist ry, b Dept . of Biology and Medical Genet ics, cSt udent of t he School of Medicine, Braće Branchet t a 20, HR- 51000 Rij eka, Croat ia E- m ail: j asm inka@m edri.hr I N TROD UCTI ON Hepat ic inj ury t hrough carbon t et rachloride ( CCl 4 ) induced lipid peroxidat ion is well known and has been ext ensively used in t he experim ent al m odels t o underst and t he cellular m echanism s behind oxidat ive dam age and furt her, t o evaluat e t he t herapeut ic pot ent ial of drugs and diet ary ant ioxidant s. Many of t hese m et hods can be used oft en t o st udy t he pot ent ial prevent ive effect of diet ary ant ioxidant s when considering t he m easurem ent s in vivo. The obj ect of t his st udy was t o observe t he effect of olive oil polyphenols on acut e liver inj ury by det erm ining t he changes in t he phospholipid ( PL FAs) and free fat t y acids ( FFAs) com posit ion, as well as t he t ot al ant ioxidant capacit y ( TEAC) of serum using t he carbon t et rachloride ( CCl 4 ) induced liver inj ury on anim al m odel. EXPERI M EN TAL Male Balb/ c m ice were divided int o 4 groups as follows: Group 1 - cont rol; Group 2 - CCl 4 t reat ed; Group 3 - CCl 4 and olive oil phenolics ext ract sim ult aneously t reat ed; Group 4 – 3- day olive oil phenolics ext ract pre- t reat ed and t hen CCl 4 t reat ed. Treat ed anim als w ere sacrificed 24 hrs aft er m ent ioned t reat m ent s; t he serum and liver were collect ed. The olive oil phenolics ext ract was previously charact erized by qualit at ive and quant it at ive evaluat ions of phenolics analysed wit h high perform ance liquid chrom at ography ( HPLC) using gallic acid as an int ernal st andard. Serum PL FAs and FFAs fract ions were isolat ed and purified by t he solid- phase- ext ract ion ( SPE) m et hod using an am inopropylsilica colum n and subsequent ly det erm ined by gas chrom at ography ( GC) . Tot al ant ioxidant st at us in t he serum was m easured as Trolox Equivalent Ant ioxidant Capacit y ( TEAC) . I m m ediat ely aft er rem oval, t he liver t issue was fixed in 10% form aldehyde and processed for hist ological exam inat ion according to t he convent ional m et hod and st aining wit h hem at oxylin and eosin ( H&E) . Calculations and Statistical Analysis D YN AM I CS OF EX PERI M EN TS SFAs= Σ% ( 14: 0+ 16: 0+ 18: 0+ 20: 0+ 22: 0+ 24: 0) ; MUFAs= Σ% ( 14: 1+ 16: 1+ 18: 1+ 20: 1) ; PUFAs= Σ% ( PUFAn- 3 + PUFAn- 6 ) ; PUFAs n–3 = Σ% ( 20: 5n- 3+ 22: 5n- 3+ 22: 6n- 3) ; PUFAs n–6 = Σ% ( 18: 2n- 6+ 18: 3n- 6+ 20: 2n- 6+ 20: 3n- 6+ 20: 4n- 6+ 22: 4n- 6) ; D9 C18 index = 18: 1n- 9/ 18: 03; D6D index = [ ( 18: 3n- 6+ 20: 3n- 6) / 18: 2n- 6] 1 ; D5D index = [ ( 20: 4n- 6/ ( 20: 3n- 6) ] 1 . ACL= [ ( Σ% Tot al FAn x n) ] / 100 ( n= carbon at om num ber) 2 ; DBI = Σ % of unsat urat ed FAs x num ber of duble bonds of each unsat urat ed FAs2 ; PI = [ ( % Monoenoic x 0.025) + ( % Dienoic x 1) + ( % Trienoic x 2) + ( % Tet raenoic x 4) + ( % Pent aenoic x 6) + ( % Hexaenoic x 8) ] 2 ; Dat a ar e r eport ed as m ean ± S.D. St at ist ical significance was assum ed wit h p< 0.05. All st at ist ical analyses w er e conduct ed using t he St at ist ica soft war e package for Window s, St at Soft , I nc. ( 2005) , Version 7.1. The significance of t he differ ences was analyzed by t he Descript ive St at ist ics by Groups ( Breakdown) - Post - hoc Com parisons of Means using Scheffe t est . Gr oup 1 Gr oup 2 Gr oup 3 ST ART Ex , 2 4 h r s Liv e r a n d se r u m colle ct ion ST ART 4 t h day CCl 4 t r eat ed ( i.p.) Gr ou p 4 Ex , 2 4 h r s Liv e r a n d se r u m colle ct ion 3 day s OOP pr e- t r eat ed Gr ou p 1 - con t r ol; Gr ou p 2 - CCl 4 t r eat ed; Gr ou p 3 - CCl 4 an d oliv e oil ph en olics ex t r act sim ult an eou sly t r eat ed; Gr ou p 4 – 3 - day s oliv e oil ph en olics ex t r act pr e- t r eat ed an d t h en CCl 4 t r eat ed Ta ble 1 . Differences in liver PL FAs and FFAs profiles bet w een t he cont rol and t est ( t reat ed) groups CCl 4 = 2 m l/ k g ( 1: 1 , v / v , Oliv e oil: CCl 4 ) ( i.p.) OOP= 10 m g/ k g oliv e oil ph en olics ex t ract s ( salin e solut ion, i.p.) PL FAs Co n t r ol group 1 8 :2 n - 6 1 8 :3 n - 6 2 0 :2 n - 6 2 0 :3 n - 6 2 0 :4 n - 6 Σ n-6 2 0 :5 n - 3 2 2 :6 n - 3 Σ n-3 Σ PUFA 1 4 :0 1 6 :0 1 8 :0 CCl 4 t r e a t e d group CCl 4 a n d OOP tr eat ed g r ou p 1 4 . 8 7 ±1 .4 7 1 7 . 2 9 ±1 .1 1 * 0 . 2 2 ±0 .1 5 * 1 6 .8 9 ±1 .0 8 0 .0 0 1 . 2 9 ±0 .3 1 0 . 1 1 ±0 .0 7 0 . 5 2 ±0 .1 5 * 0 .1 3 ±0 .0 6 28.61 ± 2.66 25.89 ± 1.11 0 . 1 4 ±0 .1 0 1 2 . 3 1 ±1 .1 4 0 . 6 9 ±0 .2 0 1 7 . 0 2 ±1 .3 8 18.14 ± 1.49 1 6 . 0 7 ±1 .9 0 42.99 ± 2.67* 2 7 . 7 2 ±1 .2 9 0 . 0 8 ±0 .0 2 1 3 . 0 5 ±1 .0 6 0 . 3 6 ±0 .2 8 2 4 :0 0 .0 0 0 .4 5 ±0 . 2 8 0 . 8 0 ±0 . 5 3 0 .6 0 ±0 .1 4 * 0 . 4 7 ±0 . 0 6 * 0 . 2 9 ±0 .1 1 0 . 8 6 ±0 .8 6 1 .0 9 ±0 . 9 0 0 . 9 9 ±0 . 8 0 26.11 ± 0.86 22.81 ± 3.09* 44.69 ± 1.48 0 .1 1 ±0 .0 2 0 . 1 4 ±0 . 0 7 6 . 2 7 ±0 . 5 6 * 1 1 . 6 2 ±1 . 0 8 * 2 9 . 2 0 ±1 . 7 5 1 7 .9 5 ±0 .7 2 * 1 9 . 5 6 ±3 . 0 7 * 0 .2 2 ±0 .2 2 0.00 0 . 4 0 ±0 . 2 4 0 .0 0 0 .7 1 ±0 .2 7 * 0 .5 4 ±0 .1 9 2 .3 3 ±1 .3 6 * 6.46 ± 2.99 8.87 ± 4.98 18.11 ± 2.09* 0 . 3 7 ±0 . 3 3 2 .7 7 ±2 .4 2 * 0 . 1 1 ±0 . 3 5 0.63 ± 0.27 0.48 ± 0.38 0 .6 3 ±0 . 2 7 9 .4 3 ±3 .0 2 * 0 .1 0 ±0 .1 2 2.90 ± 2.55 21.01 ± 2.04 3 2 .6 3 ±7 .1 4 * 2 2 .4 0 ±2 .7 2 20 0.00 0 .0 0 0 .0 0 0.00 10 1 7 . 3 5 ±1 7 .2 5 0 . 6 2 ±0 .5 7 2 4 .2 1 ±4 . 0 0 0 .6 3 ±0 . 2 8 2 2 . 3 1 ±1 2 . 6 2 0 . 5 0 ±0 . 3 6 2 .0 8 ±0 .8 7 * 5 .5 7 ±3 .1 5 * 45.34 ± 0.86* 49.45 ± 2.29* 42.15 ± 16.05 61.36 ± 5.50* 63.81 ± 13.15* 62.69 ± 6.46* 1 8 :1 n - 9 9 . 3 4 ±0 .6 5 9 . 3 8 ±0 .9 6 8 .2 8 ±0 .7 8 1 3 . 4 9 ±2 . 6 4 * 3 6 . 2 2 ±1 8 .5 7 2 4 .6 5 ±7 . 1 9 1 9 . 9 0 ±1 0 . 4 1 * 1 2 .9 3 ±6 .3 0 * 2 0 :1 n - 9 0 .0 0 9.97 ± 1.14 0 .0 0 48.09 ± 15.93 0 .0 0 * 26.84 ± 11.97* 0.00* Σ MUFA 11.35 ± 0.92 1 . 8 3 ±0 .4 8 0 11.24 ± 1.37 1 .6 1 ±0 .2 6 0 .0 3 ±0 .0 3 2 . 0 4 ±1 . 1 0 15.53 ± 3.44* 1 . 9 2 ±1 .2 0 Pla sm a t ot a l ( µM ) 0 . 1 4 ±0 .1 3 3 2 . 2 3 ±4 .9 9 0 .6 9 ±0 . 5 7 31.55 ± 7.21* 2 8 .6 9 ±5 . 0 0 1 . 0 2 ±0 . 6 9 0 . 0 3 ±0 . 0 3 * 3 0 . 0 6 ±8 . 3 0 Values ar e area per cent ( m ean± SD) ; * significant ly different from t he cont r ol det er m ined by t he Descr ipt ive St at ist ics by Groups ( Br eakdown) - Post - hoc Com par isons of Means using Scheffe t est ( p< 0.05) . Abbr eviat ions: PUFA- polyunsat ur at ed fat t y acid; MUFA- m onounsat ur at ed fat t y acid; SFA- sat ur at ed fat t y acid. Ta ble 2 . Differ ences in liver PL charact erist ics based on FA profiles det erm ined in cont rol and t est groups PL Co n t r o l group PU FA/ SFA PU FA/ M U FA 2 0 :4 / 1 8 :2 2 2 :6 / 2 0 :4 n- 3 / n- 6 1 .12 ±0 .15 0 .94 ±0 .10 * CCl 4 a n d OOP treated group 0 .99 ±0 .05 * 0 .83 ±0 .06 0 .44 ±0 .06 * 0 .49 ±0 .07 * 0 .64 ±0 .07 0 .66 ±0 .08 0 .71 ±0 .09 4 .16 ±0 .59 1 .39 ±0 .11 CCl 4 t r e a t e d group 3 .89 ±0 .64 2 .11 ±0 .13 * 4 .54 ±0 .60 2 .10 ±0 .24 * CCl 4 a n d OO P pre- t reat ed group 0 .71 ±0 .09* 0 .40 ±0 .07* 2 .39 ±0 .68* 1 .86 ±0 .23* 0 .54 ±0 .09 0 .73 ±0 .10 0 .47 ±0 .05 * D6D 0 .09 ±0 .02 0 .53 ±0 .07 * D5D 1 0.1 3±2 .7 1 15 .2 8±3 .2 3* 1 4 .6 2±3 .8 8* 1 3 .4 1±1 .88 ACL 1 8.3 8±0 .1 4 1 8 .2 5±0 .1 2 1 8 .3 3±0 .0 9 1 7 .9 9±0 .07* D 9 C1 8 D BI PI 2 02 .71 ±1 3 .2 2 2 11 .00 ±1 5 .6 1 0 .04 ±0 .01 * 1 80 .96 ±1 3 .6 2* 1 85 .15 ±1 8 .4 1* 0 .04 ±0 .02 * 1 89 .31 ±9 .98 1 97 .37 ±1 5 .1 7 0 .70 ±0 .14 0 .03 ±0 .01* 1 46 .82 ±8 .60 * 1 38 .96 ±1 0.3 4* Values are area per cent ( m ean± SD) ; * significant ly different from t he cont rol det erm ined by t he Descript ive St at ist ics by Groups ( Breakdown) - Post - hoc Com par isons of Means using Scheffe t est ( p< 0.05) . Abbr eviat ions: D9 C18- delt a 9 desat ur ase index of t he 18: 0; D6D- delt a 6 desat ur ase index; D5D- delt a 5 desat ur ase index; ACL- average chain lengt h; DBI - double bond index; PI - per oxidizabilit y index. Acknowledgements This work was support ed by t he Croat ian Minist ry of Science, Proj ect No. 062- 0000000- 0221 and 062- 0982522- 0369, DI OKI ,d.d. Zagreb and The universit y of Rij eka foundat ion. 1 .0 3 ±1 .4 1 16.30 ± 6.72* 3 2 .5 2 ±1 1 .3 4 # 35 9.35 ± 5.03 4 . 6 1 ±2 . 9 6 * + 40 3 6 . 1 0 ±1 0 . 8 2 * 2 .5 4 ±0 . 3 7 * 45.77 ± 2.26* 1 . 9 1 ±0 .4 3 45 b. 3 3 .7 9 ±7 . 0 1 * 41.89 ± 2.60 1 6 :1 n - 7 50 1 6 . 8 8 ±4 .5 6 7 . 2 9 ±2 .4 2 7.09 ± 3.03 6 . 5 7 ±5 . 1 3 * a. 5 .0 4 ±3 .5 4 * 0 . 9 9 ±0 . 8 0 * 0 .0 0 9.76 ± 2.96 0 . 2 9 ±0 . 1 4 * CCl 4 a n d OOP pr etreated g r ou p 1 .1 0 ±0 . 9 0 0 . 0 5 ±0 .0 5 0.49 ± 0.32 35.01 ± 3.50* 0 . 5 1 ±0 . 3 5 4 .5 0 ±2 . 5 7 * 0 . 4 3 ±0 .2 5 12.21 ± 1.08* 0 .4 3 ±0 . 2 1 6 . 4 6 ±2 .6 5 9.27 ± 2.86 0 . 2 9 ±0 . 1 2 * 2 7 .0 6 ±1 .0 7 0 . 0 5 ±0 .0 5 0 .0 0 0 . 2 1 ±0 .0 8 1 7 .4 2 ±1 .5 3 1 7 . 8 9 ±1 .1 4 * CCl 4 a n d O O P t r e a t e d g r ou p 1 . 4 8 ±0 .5 1 18.58 ± 1.83 0 . 1 1 ±0 .0 2 CCl 4 t r e a t e d g r ou p 0 .0 0 0 .6 7 ±0 .2 2 17.01 ± 1.91 Co n t r ol group 1 5 . 9 2 ±2 . 6 7 8 .3 2 ±0 .8 2 * 0 . 7 2 ±0 .3 0 2 8 . 4 0 ±3 .1 6 CCl 4 a n d OOP pretreated group 0 .1 5 ±0 .1 5 * 7 . 6 2 ±0 .6 3 * 46.76 ± 3.24 2 0 :0 Σ SFA FFAs m M Trolox Fa t t y a cid s * 30 25 15 1 2 3 4 Gro u p Figu r e 1 . a . Hist ological analysis of t he liver from m ale Balb/ c m ice. H&Est ained liver sect ions from : A- norm al, B- CCl 4 t reat ed, C- CCl 4 and OOP sim ult aneously t r eat ed and D - 3- days OOP pre- t reat ed and t hen CCl 4 t reat ed m ice, m agnificat ion = 400X; b. Serum t ot al ant ioxidat ive capacit y ( TAC) * significant ly different from t he cont rol and group 2, + from t he group 2 and 3, # from t he group 2 and 4, det erm ined by t he Descript ive St at ist ics by Groups ( Breakdown) - Post - hoc Com parisons of Means using Scheffe t est ( p< 0.05) . CON CLUSI ON S The concent rat ions of PL FAs and FFAs were alt ered by CCl 4 adm inist rat ion, as well as by t he sim ult aneous use of olive oil phenolics wit h CCl 4 . Significant differences in PL FAs were not ed in t he liver t issue aft er t he adm inist rat ion of CCl 4 . The percent cont ribut ion of liver PL FAs in group 2 as com pared t o t he cont rol group was significant ly higher for 18: 0, 18: 2n- 6, 18: 3n- 6 and sat urat ed fat t y acids ( SFA) , and lower for 20: 3n- 6, 20: 4n- 6 and polyunsat urat ed fat t y acids ( PUFAs) . I n t he liver FFAs fract ion, percent cont ribut ion was significant ly lower as com pared t o t he cont rol group for 14: 0, 20: 4n- 6, 20: 1n- 9 and m onounsat urat ed fat t y acids ( MUFAs) , w hile significant ly increased for 16: 0 and sat urat ed fat t y acids ( SFAs) . Com pared t o t he CCl4 t reat ed group, olive oil ant ioxidant s group showed a reduct ion in t he 18: 1n- 9. Our experim ent al st udy showed t hat pre- t reat m ent wit h olive oil phenolics result ed in m ore fat t y acid changes in t he liver. Com pared t o CCl 4 t reat ed group, significant changes in 14: 0, 18: 1n- 9, 20: 4n- 6 in t he PL FAs fract ion, and 14: 0 in t he FFAs fract ion were found. Maj or changes were not ed in bot h lipid liver fract ions com pared t o group 3 and 4. So, com parison bet ween pre- t reat ed and t reat ed olive oil phenolics group showed significant differences in 14: 0, 16: 0, 18: 1n- 9, 18: 3n- 6, 20: 3n- 6, 20: 4n- 6, 20: 5n- 3 and 22: 6n- 6 in t he PL FAs fract ion and 14: 0, 18: 2, 20: 3n- 6, 24: 0 and 22: 6n- 3 in t he FFAs fract ion. CCl 4 - t reat ed groups showed t hat t ot al ant ioxidant capacit y was higher com pared t o t he cont rol. Liver hist opat hology indicat ed t hat olive oil polyphenols reduced t he inj ury score of fat t y degenerat ion incidence and liver lesions induced by CCl 4 in m ice. References 1. Zuij dgeest - Van Leeuwen SD, Van der Heij den MS, Riet v eld T, Van der Berg JWO, Tilt anus HW, Burger s JA, Wilson JHP, Dagnelie PC ( 2002) Br J Cancer 87: 1370 – 1378 2. Pam plona R, Por t ero- Ot ín M, Riba D, Ruiz C, Prat J, Bellm unt MJ and Barj a G ( 1998) J Lipid Res 39: 1989–1994