INTRODUCTION  

Phytosterols  or  plant  sterols  are  a  group of  steroid  alcohols,  phytochemicals naturally occuring in plants. They have a chemical  structure  which  is  similar  to cholesterol  (Weihrauch  &  Gardner  1978) and exist in several forms in plants (Law 2000; Katan et al. 2003; Abumweis et al. 2007) including β-sitosterol, campesterol, stigmasterol  and  cycloartenol  (Ostlund 2002).  Phytosterols  are  natural  compo- nents  found  in  the  human  diet.  It  made up  0.1  to  0.5%  w/w  of  vegetable  oils  or margerine  (Kochlar  1983)  and  are  also found in corn, wheat and rice. Phytosterol intake varies according to the type of diet taken.  Europeans  consume  about  200- 300  mg/day  of  phytosterol  (Morton  et  al. 1995)  while  the  vegetarian  Japanese have  a  higher  intake  of  phytosterol  (Nair et al. 1984). 

Phytosterols  are  well  known  for  their ability  to lower  plasma  cholesterol  level (Klingberg et al. 2008) by interfering with the  absorption  of  cholesterol  from  the gastrointestinal  system  (Jones  1999; Hayes  et al.  2004;  Jia  et al.  2007).

Phytosterols  have  also  been  shown  to possess  anti-cancer  properties  against gastric,  colon,  ovary  and  breast  cancers (Choi  et al. 2007;  Mendilaharsu  et al. 1998; De Stefani et al. 2000; McCann et al.  2003;  Ju  et  al.  2004).  These  studies suggested  that phytosterols  may  be useful in the prevention of cardiovascular diseases and cancers.

Oxidative  stress  is  thought  to be  in- volved  in the  pathogenesis  of coronary and peripheral arterial diseases (Smith et al. 1993). During lipid peroxidation, lipids are  oxidized  to  form  free  radicals  which can  cause  extensive  tissue  damage. Several  cardiovascular  risk  factors  have been  identified  that  promote  cardiovas- cular  disease  via  lipid  peroxidation (Rumley et al. 2004). Therefore, reducing lipid  peroxidation  is  important  in preven- tion  of  cardiovascular  disease.  This  may be  achieved  through  the  anti-oxidant properties  of  phytosterols.  It is  also  be- lieved  that  oxidative  stress  may  contri- bute  to  carcinogenesis  by  DNA.  How- ever, there is a growing body of evidence showing  that lipid  peroxidation  do not promote carcinogenesis but may actually inhibit  most  cancer  cells  (Zanetti  et  al. 2003; Gago-Dominguez et al. 2005). 

Phytosterols  were  found  to  exert  anti- oxidant effects on the oxidation of methyl linoleate  in  solution.  They  also  sup- pressed  the  oxidation  and  consumption of  α-tocopherol  in  β-linoleoyl-γ-palmitoyl phosphatidylcholine  (PLPC)  liposomal membranes  (Yoshida  &  Niki  2003).  Vi- vancos and Moreno (2005) reported that phytosterols  were  able  to  increase  the anti-oxidant  enzyme  activities  of  supe- roxide  dismutase  and  glutathione  perox- idase  in  cultured  macrophage  cells  ex- posed  to  oxidative  stress  by  phorbol  12- myristate 13-acetate. Therefore, an alter- native  mechanism  of  protection  from oxidative  stress  by  phytosterols  is  by  in- creasing  the  antioxidant  enzyme  activities.

There were limited published reports on the  effects  of  phytosterol  on  lipid  peroxi- dation.  The  aim  of  our  study  is  to  deter- mine  the  effects  of  pretreatment  with phytosterol  on  lipid  peroxidation  in plasma  and  organs  of  rats  exposed  to carbon tetrachloride.  

MATERIALS AND METHODS 

A  preliminary  study  was  conducted  to determine  the  suitable  dose  of  carbon tetrachloride  which  can  induce  lipid  pe- roxidation  in  various  rat  organs.  Hafsah (2005) found that the dose of 1.0 ml/kg of carbon  tetrachloride overwhelmed  the effects  of  phytosterols  which  were  given at  weekly  doses  of  140  mg/kg  (unpublished). 

In  this  study,  24 male  Sprague-Dawley rats weighing between 175 to 200 grams were  obtained  from  the  UKM  Animal House.  The  rats  were  housed  in plastic cages at room temperature (29±3ºC) and daily  dark/light  cycle.  The  rats  were  fed standard rat pellets (Gold Coin, Malaysia) and  distilled  water  ad libitum.  The  rats were  allowed  to adjust  to the new  envi- ronment for a week before the study was started.  The  study  was  approved  by  the UKM Animal Ethics Committee.

The  rats  were  randomly  divided  into  4 groups  of  normal  control  (NC),  carbon tetrachloride  (CCl4),  phytosterol  (P)  and phytosterol+carbon  tetrachloride (P+CCl4).  The  latter  two  groups  were pretreated  with  a subcutaneous  injection of  phytosterol  (MPOB,  Malaysia)  at a dose of 140 mg/kg, once a week for five weeks  (Yoshida  &  Niki  2003).  These phytosterols were extracted from palm oil and  are  composed  of  60%  β-sitosterol and  40%  stigmasterol  and  campesterol. The  NC  and  CCl4  groups  received  2 ml/kg  olive oil  (vehicle)  (Oomerbhoy  Ltd, Mumbai)  subcutaneously  once  a  week for  the same  duration.  Rats  in the  CCl4 and  P + CCl4  groups  were  then given  a single dose of 0.5 ml/kg carbon tetrachlo- ride (BDH Chemicals, England) diluted in olive  oil  via  oral  gavage  to  induce  lipid peroxidation.  Rats  in the  NC  and  P groups  received  equivalent  amounts  of olive  oil  (vehicle).  After  24  hours,  blood samples  were  taken  via the  orbital  sinus under anesthesia. The blood  was left for 30  minutes  and  centrifuged  at 3000  rpm for  5 minutes  to  separate  the  plasma. The  plasma  was  stored  at -20°C.  The rats  were  then  sacrificed  and the  liver, heart,  kidneys  and  lungs  were  dissected out.  The  plasma  and  tissue  ma- londialdehyde  (MDA)  levels  were  meas- ured  according  to  the  method  of  Led- wozyw  et al.  (1986).  According  to this method,  malondialdehyde  is  formed  as an end product of lipid peroxidation which reacts  with  TBA  (Thiobarbutaric  acid) reagent  under  acidic  condition  to gener- ate a pink-coloured product. Plasma (0.1 ml) was added to 0.4 ml of distilled water, followed  by  the  addition  of  2.5 ml  of trichloroacetic  acid  (TCA).  The  mixture was  left  at room  temperature  for  15 mi- nutes. TBA (1.5 ml) was then added and heated  in a water  bath  at  100°C  for  30 minutes  until  a faint  pink  colour  was  ob- tained.  After  cooling,  the colour  was  extracted in 1 ml of buthanol and the inten- sity  was  measured  using  the  spectro- photometer  at  EX  515nm  and  EM 553nm.  1,1,1,3-tetraethoxypropane (Sigma, USA) was used as the standard.    The  results are expressed as  mean±SEM. The statistical significance of the  data  was  determined  using  one-way analysis  of  variance  (ANOVA)  and  post hoc  Tukey  test.  The  level  of  significance was set at p<0.05. 

RESULTS 

In order to determine the suitable dose of carbon  tetrachloride  for  induction  of  lipid peroxidation,  we  have  carried  out a pre- liminary  study.  In the study,  rats  were given  either  0.5 ml/kg  or  0.75 ml/kg  of carbon  tetrachloride  and  sacrificed  after 24  hours.  The  plasma  and liver  melon- dialdehyde  (MDA)  were  then  measured. We found from our preliminary study that the  lowest  dose  of  carbon  tetrachloride that  would  produce  significant  lipid  pe- roxidation  in  plasma  and liver  was  0.5 ml/kg  (Figures  1 and  2).  Results  of  the present  study  showed  that  the  plasma MDA of carbon tetrachloride (CCl4) group was  significantly  higher  than  the  normal control group. The plasma MDA levels of the  P and P+CCl4  groups  were  signifi- cantly lower than the CCl4 group (Figure 3).  The  hepatic  MDA  level  for  the  CCl4 group  was  significantly  higher  than  the normal  control  group.  The  hepatic  MDA levels of the P and P+CCl4  groups  were significantly  lower  than  the  CCl4  groups. In  addition,  the  hepatic  MDA  level  of  P group  was  significantly  lower  than the normal  control  group  (Figure  4).  There was  no significant  change  in renal  MDA level  in  the  CCl4  group  compared  to the normal  control  group.  The  renal  MDA levels for the P and P+CCl4 groups were significantly  lower  compared  to the CCl4 group  (Figure  5).  There  was  no  signifi- cant  change  in the  cardiac  MDA  level  in the CCl4 group compared to normal control  group. The  cardiac MDA  level for  the P group was significantly lower than P and P+CCl4   groups  (Figure  6).  The  MDA  le- vels  in  the  lungs  showed  no  significant findings in all the groups (Figure 7). 

DISCUSSION

Carbon  tetrachloride  is  metabolised  by cytochrome P-450 enzymes in the liver to the  reactive  trichloromethyl  radical.  The radical  is  oxidized  further,  forming  the even  more  reactive  trichloromethylpe- roxyl radical (McGregor & Lang 1996;   IPCS  1999).  These  reactive  metabolic intermediates  of  carbon  tetrachloride, particularly trichloromethylperoxyl radical, can cause lipid peroxidation (IPCS 1999). In  our  study,  carbon  tetrachloride  has induced  lipid  peroxidation  as  shown  by elevations  of  MDA  levels  in  the  rat’s plasma,  liver,  kidneys,  heart  and  lungs. However,  only  the  plasma  and  hepatic MDA  levels  were  significantly  raised. to  cause injuries  to  various  organs  of  the  body (Reynolds et al. 1984) including the liver, kidneys,  heart,  lungs,  gastrointestinal tract  and  central  nervous  system (ATSDR  2005).  The  primary  targets  for carbon  tetrachloride  toxicity  are  the  liver and kidneys (IPCS 1999).

Phytosterols taken into the body are in- corporated into cell membranes (Awad et al.  2004)  and  are  highly  concentrated  in the  lungs,  adrenal  cortex,  intestinal  epithelia and  ovaries (Sanders et al.  2000). In our study, we find that phytosterol pre- treatment  was  able  to prevent  elevation of  MDA  levels  in the  plasma,  liver  and kidneys  but  was  unable  to  do  the  same for  the heart  and  lungs.  Therefore,  pretreatment  with  phytosterol  was  able  to reduce  lipid  peroxidation  in  the  plasma and protect the liver and kidneys against lipid  peroxidation  damage.  These  find- ings  were  consistent  with  other  findings that showed that pretreatment  with other antioxidants,  such  as  vitamin  E reduced the  hepatotoxic  action  of  carbon tetrach- loride (IPCS 1999). 

In  a study  by  Mora-Ranjeva  et al. (2006), phytosterol in the form of sitoste- rol  and  stigmasterol  were  incorporated into  human  keratinocytes  (SVK14  line) and  exposed  to ultraviolet  light.  It was found  that sitosterol  induced  significant decrease  (-30%)  in  lipid  peroxidation whereas stigmasterol markedly increased lipid peroxidation (+70%). Results of this  study  also  showed  that  the  effects  of phytosterol on lipid peroxidation also de- pended on the form of the phytosterol. In this  study,  we have  used  phytosterols derived from palm oil which was made up of 60% β-sitosterol and 40% campesterol and stigmasterol.

We  found  that  there  were  measurable MDA  levels in the plasma and organs of rats  in the normal  control  group.  This may  be  contributed  by  the  free  radicals being  produced  even  under  normal  con- ditions  by  either  leakage  of  activated oxygen  from  mitochondria  during  oxida- tive  phosphorylation  or  by  the  multiple redox-active  flavoproteins  (Messner  & Imlay  2002;  Imlay  2003;  Seaver  &  Imlay 2004).  Phytosterol supplementation was able to reduce the MDA levels of the liver and  heart  of  normal  rats.  Based  on this result, phytosterol may be useful as sup- plements for  healthy  individuals  to main- tain their oxidative status but this requires further studies. 

The  phytosterol  dosage  used  in  this study is equivalent to the human intake of 20  mg/kg  body  weight/day.  This  is  high considering  that  the  daily  intake  of  phy- tosterols in the human diet is about 2.5 to 4.0  mg/kg  bodyweight  (Morton  et al. 1995).  With  regards  to its  toxicity,  ad-

ministration  of  phytosterols  at the  daily doses  of  6.6 g/kg  bodyweight  in rats  did not show any subchronic toxicity or tera- togenic effects (Hepburn et al. 1999). In a human  study,  the  daily  intake  of  9.0 grams of phytosterols for eight weeks did not  cause  any  adverse  effects  (Katan  et al.  2003).  However,  it  was  shown  to  re- duce  absorption  of  lipid  soluble  antioxi- dants  such  as  α-carotene,  β-carotene and vitamin E (Law 2000).

In  conclusion,  phytosterols  may  be used  to  reduce  lipid  peroxidation  in  the plasma,  liver  and  kidneys.  Phytosterol supplementation  has  a  good  safety  pro- file  and  may  further  reduce  lipid  peroxi- dation in healthy individuals. 

ACKNOWLEDGEMENTS 

We  would  like  to  thank  the  staff  of  the Pharmacology Department for their tech- nical assistance.