during the last 50 years nearly 1000 drugs have been identified as possible causes of hepatic injury , termed drug - induced hepatotoxicity , especially adriamycin ( adr ) . cancer is one of the most of common causes of death in the world , and the prevalence of adr - induced hepatotoxicity , therefore , is increasing . while adr is the most effective drug in cancer tissue , it has , unfortunately , some adverse effects in noncancerous tissue , including liver , heart , kidney , brain , immune system , and testis . one reason may be that the liver is the primary involved in detoxification of exogenous and endogenous toxins , especially toxic chemicals , probably resulting in hepatotoxicity . another reason is that reactive oxygen species ( ros ) are related toinvolved in adverse effects in normal liver tissue . multiple studies have reported that adr s toxic effects could relate to generating oxidative stress in the normal liver ; therefore , some antioxidants ( e.g. , selenium , vitamin e , and nac ) can decrease adr - induced liver damage . also , it has been well documented that adr drastically increases lipid oxidation and mitochondrial ros content , and decreases liver antioxidant enzymes and mitochondrial function . other factors contributing to organ toxicity include adr generation of the inflammatory cascade , and , eventually , programmed cellular death ( apoptosis ) . to enhance the therapeutic effects and decrease the toxic adverse effects of adr , it is essential to identify and characterize the mechanisms by which these effects occur in the liver . adr s hepatotoxicity has been shown to be associated with oxidative stress linked to redox - cycling of the drug . the mechanism of generation of oxidative stress induced by adr in liver tissue has not been completely defined . one is that the adr redox - cycling may be triggered by 1-electron reduction with the formation of adr radical . the reaction is catalyzed by various enzymes ( e.g. , nadph - cytochrome p450 reductases , nos , nadph oxidase , and catalase ) . although reoxygenation is needed to convert it from its radical to its nonradical form , the superoxide radical is simultaneously manufactured . moreover , those enzymes caused in adr radical production have been shown to be highly concentrated in hepatocytes . adr radicals delocalized fe adr from ferritin and generate h2o2 , eventually causing oxidant injury in the liver . the renin - angiotensin system is frequently activated in patients with chronic liver diseases , such as cirrhosis ( which contributes to fibrosis progression ) , hepatocellular carcinoma [ 810 ] , and liver inflammation . angiotensin ii ( ang - ii ) is an important effector peptide of this system . ang - ii has some crucial physiological functions , such as cell contraction , cell growth / hypertrophy , apoptosis , differentiation , and secretion of inflammatory cytokines . ang - ii actions are mainly mediated by its 2 sub - receptors  the ang - ii type 1 receptor ( at-1 ) and the ang - ii type 2 receptor ( at-2 ) . these 2 subunits mediate the effects of ang - ii on various organs , and at-2 ( mostly in fetal cells ) is less common than at-1 . increased systemic ang - ii was reported to augment and promote inflammation and oxidative stress . in target cells , ang - ii induces free radical formation and oxidative stress , stimulating redox - sensitive intracellular pathways . in addition to systemic the ras , virtually every organ system in the human body has been shown to possess a local ang signaling system in peripheral tissues ( e.g. , liver , vasculature , kidneys , adrenal glands , heart , and immune cells . the systemic ras is activated in patients with cirrhosis and the local ras is induced in fibrotic livers and activated hepatic stellate cells ( hscs ) . it has been shown that ang - ii might modulate mitochondrial membrane potential and transcription of respiratory chain subunits , and may stimulate the production of ros in mitochondria , thus leading to mitochondrial failure in cardiac , renal , and vascular smooth muscle cells . mitochondrial failure probably causes the massive production of ros . according to current understanding of adr and ras , adr might crosstalk with the local ras in liver tissue to amplify its adverse effects . the hypothesis in the present study , therefore , was that adr cooperates with the local ras in the liver tissue to generate free radical formation , resulting in oxidative stress and attenuating mitochondrial function . hence , the aim of the study was to investigate the potential prophylactic effects of attenuated angiotensin - ii production in the liver against mitochondrial dysfunction in rats with adr - induced oxidative stress . all experimental procedures were carried out in strict accordance with the animal experimentation local ethics committee of the erciyes university . thirty - five male sprague - dawley rats were randomly divided into 5 treatment groups as shown figure 1 : control ( n=7 ) , adr ( n=7 ) , cap ( captopril + adr , n=7 ) , al ( aliskiren + adr , n=7 ) , and cap + al ( captopril + aliskiren + adr , n=7 ) . adr ( adriamycin hcl , adriblastina vial 10 mg , pharmacia ) was administered in 4 equal injections ( each intraperitoneal injection containing 4 mg / kg in saline , every other day , to reach a total dose of 16 mg / kg ) during 7 days . on the same day the control group received the same volume of physiological saline . captopril was intragastrically administered ( 10 mg / kg , every day for 7 days ) . aliskiren was intragastrically administered ( 50 mg / kg , every day for 7 days ; figure 1 ) . after the last drug treatment , liver tissues were collected , and then kept at 80 c until use ; cytosol and mitochondria were separated by centrifugation for use in later biochemical assays . the fluorescent mitochondrial - specific cationic dye 5,5,6,6-tetrachloro-1,1,3,3-tetraethyl benzimidazolylcarbocyanine iodide ( jc-1 ) was used for assessment of mmp according to the manufacturer s protocol . to measure atp content and adr - toxicity in the liver , the kit is based upon the bioluminescence of atp that is present in all metabolically active cells . total antioxidant status ( tas ) of the liver s cytosol and mitochondria were measured according to manufacturer s protocol based on colorimetric measurement ( rel assay kit , turkey ) . the results are expressed in mmol trolox equiv./l . total oxidant status ( tos ) of the liver s cytosol and mitochondria were measured using a rel assay kit ( rel assay kit , turkey ) , based on colorimetric measurement . the tos - to - tas ratio was used as the oxidative stress index ( osi ) . the osi value was calculated as follows : osi = ( tos)/(tas ) ( mol / l/ mol trolox equivalent / l ) . comparison among different groups were made using multiple analyzes of variance ( anova ) , followed by a post hoc protected tukey test p<0.05 was considered to be significant . all experimental procedures were carried out in strict accordance with the animal experimentation local ethics committee of the erciyes university . thirty - five male sprague - dawley rats were randomly divided into 5 treatment groups as shown figure 1 : control ( n=7 ) , adr ( n=7 ) , cap ( captopril + adr , n=7 ) , al ( aliskiren + adr , n=7 ) , and cap + al ( captopril + aliskiren + adr , n=7 ) . adr ( adriamycin hcl , adriblastina vial 10 mg , pharmacia ) was administered in 4 equal injections ( each intraperitoneal injection containing 4 mg / kg in saline , every other day , to reach a total dose of 16 mg / kg ) during 7 days . on the same day the control group received the same volume of physiological saline . captopril was intragastrically administered ( 10 mg / kg , every day for 7 days ) . aliskiren was intragastrically administered ( 50 mg / kg , every day for 7 days ; figure 1 ) . after the last drug treatment , liver tissues were collected , and then kept at 80 c until use ; cytosol and mitochondria were separated by centrifugation for use in later biochemical assays . the fluorescent mitochondrial - specific cationic dye 5,5,6,6-tetrachloro-1,1,3,3-tetraethyl benzimidazolylcarbocyanine iodide ( jc-1 ) was used for assessment of mmp according to the manufacturer s protocol . to measure atp content and adr - toxicity in the liver , a bioluminescent kit was used according to the manufacturer s protocol . the kit is based upon the bioluminescence of atp that is present in all metabolically active cells . total antioxidant status ( tas ) of the liver s cytosol and mitochondria were measured according to manufacturer s protocol based on colorimetric measurement ( rel assay kit , turkey ) . the results are expressed in mmol trolox equiv./l . total oxidant status ( tos ) of the liver s cytosol and mitochondria were measured using a rel assay kit ( rel assay kit , turkey ) , based on colorimetric measurement . the tos - to - tas ratio was used as the oxidative stress index ( osi ) . the osi value was calculated as follows : osi = ( tos)/(tas ) ( mol / l/ mol trolox total antioxidant status ( tas ) of the liver s cytosol and mitochondria were measured according to manufacturer s protocol based on colorimetric measurement ( rel assay kit , turkey ) . total oxidant status ( tos ) of the liver s cytosol and mitochondria were measured using a rel assay kit ( rel assay kit , turkey ) , based on colorimetric measurement . the tos - to - tas ratio was used as the oxidative stress index ( osi ) . the osi value was calculated as follows : osi = ( tos)/(tas ) ( mol / l/ mol trolox comparison among different groups were made using multiple analyzes of variance ( anova ) , followed by a post hoc protected tukey test p<0.05 was considered to be significant . adr - induced toxicity is well known to be associated with increased oxidative stress , resulting from increased levels of reactive oxygen species and decreased levels of antioxidants . they were measured as the type of mitochondria and cytosol from liver tissue in the present study . mitochondrial and cytosolic tos were significantly increased by adr compared to cont ( p<0.05 ; figure 2a , 2b ) . the inhibition of ang - ii in the liver was restored to mitochondrial at cap and cytosolic tos at cap and cap + al , ( p<0.05 ; figure 2a , 2b ) . the elevation of tos in mitochondria and cytosol could be related to ang - ii production in liver tissue . adr decreases tas in mitochondria and cytosol , but not significantly compared to the control group . the inhibition of ang - ii tended to increase of all groups , but not significantly ( figure 3a , 3b ) . the level of oxidative stress was calculated by using the ratio of tos to tas in mitochondria and cytosol . osi in mitochondria and cytosol were significantly elevated in the adr group ( p<0.05 vs. cont ; figure 4a , 4b ) . however , the inhibition of local ang - ii production by renin and/or ace inhibitors led to a significant decrease in mitochondrial osi at cap and cap + al groups and cytosolic osi at all groups , resulting from decreasing tos ( p<0.05 vs. adr group , figure 4a , 4b ) . mmp was estimated via the ratio of red to green fluorescence by using a special mitochondrial cationic dye ( jc-1 ) to demonstrate adr s toxicity in mitochondria . adr caused sharp dissipation of mmp ( p<0.05 vs. adr group ; figure 5 ) . in parallel , adr s effect on mmp significantly decreased atp production ( p<0.05 vs adr group ; figure 6 ) . however , attenuation of local ang - ii production by renin and/or ace inhibitors significantly increased atp production in liver mitochondria ( p<0.05 ; figure 6 ) . these data demonstrate that angiotensin - ii might be able to cooperate with adr to derive oxidative , stress causing reduced mitochondrial membrane potential , resulting in minimizing atp production in liver tissue . adr - induced toxicity is well known to be associated with increased oxidative stress , resulting from increased levels of reactive oxygen species and decreased levels of antioxidants . they were measured as the type of mitochondria and cytosol from liver tissue in the present study . mitochondrial and cytosolic tos were significantly increased by adr compared to cont ( p<0.05 ; figure 2a , 2b ) . the inhibition of ang - ii in the liver was restored to mitochondrial at cap and cytosolic tos at cap and cap + al , ( p<0.05 ; figure 2a , 2b ) . the elevation of tos in mitochondria and cytosol could be related to ang - ii production in liver tissue . adr decreases tas in mitochondria and cytosol , but not significantly compared to the control group . the inhibition of ang - ii tended to increase of all groups , but not significantly ( figure 3a , 3b ) . the level of oxidative stress was calculated by using the ratio of tos to tas in mitochondria and cytosol . osi in mitochondria and cytosol were significantly elevated in the adr group ( p<0.05 vs. cont ; figure 4a , 4b ) . however , the inhibition of local ang - ii production by renin and/or ace inhibitors led to a significant decrease in mitochondrial osi at cap and cap + al groups and cytosolic osi at all groups , resulting from decreasing tos ( p<0.05 vs. adr group , figure 4a , 4b ) . mmp was estimated via the ratio of red to green fluorescence by using a special mitochondrial cationic dye ( jc-1 ) to demonstrate adr s toxicity in mitochondria . adr caused sharp dissipation of mmp ( p<0.05 vs. adr group ; figure 5 ) . in parallel , adr s effect on mmp significantly decreased atp production ( p<0.05 vs adr group ; figure 6 ) . however , attenuation of local ang - ii production by renin and/or ace inhibitors significantly increased atp production in liver mitochondria ( p<0.05 ; figure 6 ) . these data demonstrate that angiotensin - ii might be able to cooperate with adr to derive oxidative , stress causing reduced mitochondrial membrane potential , resulting in minimizing atp production in liver tissue . because both adr and ang - ii play important roles in oxidative stress related to some liver pathologies , we hypothesized that adr may cause liver damage via local ang - ii production . several lines of evidence from our current study support this hypothesis : inhibition of ang - ii production attenuates several markers of adr - induced liver injury and decreases tos , resulting in decreased oxidative stress and increased mmp and atp in rats exposed to adr . these results suggest that ang - ii cooperates with adr s hepatotoxicity in rats , confirming the potential health benefit of inhibition of ang - ii in vivo against oxidative stress induced by adr . it is clear that adr causes irreversible liver damage , triggering apoptotic processes in normal liver tissue . adr - mediated hepatotoxicity has been shown to include focal damage in hepatocytes , vascular damage , and steatosis . thus , its hepatotoxicity was ascribed to subcellular hepatic alterations , including polymorphic mitochondria and cytoplasmic vacuolization . various compounds ( most of them antioxidants , such as vitamin e , erdosteine , cystathionine , ator , and catechin in adr - induced hepatotoxicity ) have been reported to prevent liver injury induced by oxidative stress . recent studies have been focused on better understanding oxidative stress mediated by adr s toxicity and the outcome of adverse effects in heart , liver , and kidney . during the adr redox - cycling process , adr gains an electron and is reduced into adr radical - catalyzed by several endogenous enzymes , including p-450 . so , it was not so surprisingly that the liver has more p-450 enzyme than other tissues due to its detoxification activity . then , adr shifts to its original form by re - oxygenation , generating reactive oxygen radicals . eventually , these radicals promote oxidative stress through altering the redox status of essential proteins . in agreement with this , a previous study reported a significant increase in mda levels , which is a good indicator of oxidative damage . the same study also showed significant attenuation of some antioxidant levels ( e.g. , sod , gsh , and gsh - px ) in rat liver treated with adr . another study reported that adr increased ros concentration and oxidative stress biomarkers , as well as decreasing antioxidant enzyme activities and mitochondrial function in the liver . as shown figures 24 , all results are consistent with the current study s results . redox cycling occurs in the cytoplasm , endoplasmic reticulum , and ( especially ) in mitochondria . the compartmentalization of these kinds of subcellular oxidative stress is important for cell functions , especially because mitochondrial oxidative stress can destroy mitochondrial function . we separately measured oxidative stress ( osi , tos , and tas ) in cytosol and mitochondria compartments in the present study , because oxidant radicals may freely cross intracellular membranes ; therefore , subcellular compartmentalization of antioxidants may significantly modulate the harmful activity of ros in subcellular compartments . therefore , the level of mitochondrial oxidative stress is probably more important than the oxidative stress in all other cell organelles . according to this assumption therefore , the deficiency of mitochondrial antioxidant defense might increase the release of oxidant from the mitochondrial matrix , which can consequently oxidize some cytoplasmic proteins and cell signaling , as well as membrane pumps proteins . moreover , the disruption of the mitochondrial membrane potential also is important in regulating necrosis and apoptosis . our results show that the mitochondria are the dominant source of oxidative stress - induced by adr in the liver . mitochondrial dysfunction associated with adr - related toxicities might cause the observed change in activities of typical markers of oxidative status in damaged liver tissue . consequently , as an important source of ros production , mitochondria could also be a primary target susceptible to ros attacks . the defects in the mitochondrial structural design would lead to the adaptation of mitochondrial metabolism , resulting in decreased activity of mitochondrial enzymes in the liver exposed to adr , hence becoming a principal contributor to intrinsic cell dysfunction . free radicals also stimulate the pro - apoptotic protein ( e.g. , bax ) , resulting in loss of hepatocyte . the other reason hepatocytes are reduced by adr is associated with redox alteration , causing irreversible liver toxicity and its changes are reported to be important in regulating hepatocyte division . the changing of redox potential may be common attributing for cells division the fact that ros have been suggested to modulate the cell cycle in many cell lines . loss of hepatocytes probably leads to even worse liver toxicity of adr , although it has been repored that some aspect of angiotensin signaling inhibition could be involved in alleviating adriamycin - induced cardiomyopathy   and renopathy . alterations in ras have frequently been implicated in the pathophysiology of various diseases involving the heart , lung , kidney , and liver [ 10,2931 ] . ang - ii , an essential member of the ras , handles both physiologic   and pathophysiologic   effects of ras . it is associated with liver fibrogenesis , cirrhosis , portal hypertension , and hepatic ischemia / reperfusion injury . ang - ii leads to apoptosis , tumor vessel growth , tumor invasion and metastasis , and immunosuppression , leading to the development of tumors . in the other words furthermore , recent studies have reported that at-1 overexpression may be associated with hepatocellular , non - small cell lung , gastric , breast , ovarian , bladder , pancreatic , and prostate cancers . evidence shows that ang - ii plays a role in liver inflammation in humans and rodents ( 11 ) . the importance of ras has been shown in development of hepatic fibrosis , and the benefits of modulation of ace gene expression have been reported . cap , an angiotensin - converting enzyme blocker , was able to attenuate growth in a murine model of colorectal cancer liver metastases . this so - called local or tissue ras has various roles , such as the promotion of inflammation and fibrosis . a recent study   reported that local ras is present in the liver , heart , kidney , lacrimal gland , and lung . each kind of ras expresses itself not only in the fibrotic liver , but also in liver parenchymal cells , including hepatocytes . this local angiotensin is implicated in fibrotic pathogenesis in the liver . also , activated at-1 expresses on the surface of hscs and assembles into the hepatic fibrotic area . local or tissue ras might be highly related to systemic ras under pathophysiologic situations , or independent of the circulation system . renin and ace inhibitors  to prevent the local ras , for evaluation of the relationship between adr s hepatotoxic actions and ang - ii . however , there are limited studies on neurohormonal activation of adr on tissues . to the best of our knowledge , there is no published data on the association of adr and ang - ii in liver mitochondrial dysfunction . hence , it is not easy to explain the physiologic relevance or the underlying mechanism of the present observations ; these deserve attention in future studies . however , previous research has suggested that increased intracellular ang - ii in diabetic rat heart was associated with enhanced cardiac myocyte apoptosis , fibrosis , and oxidative stress . the latter conditions were alleviated better by a renin inhibitor , which blocks the intracellular ras , compared to ang - ii receptors and ace inhibitors . the renin inhibitor , aliskiren , was reported to enter the cell and inhibit the intracellular production of angiotensin ii . the above findings suggest similar partitioning of aliskiren in the heart , which might have resulted in sufficiently high intracellular levels of aliskiren to inhibit rat renin in our studies . it is reported that a renin inhibitor prevents both intracellular and extracellular ang - ii synthesis . consistent with our previous study , we observed that aliskiren had some effective impacts on preventing oxidative stress in kidney - exposed to adr s toxicity . the same study also reported attenuated progression of hepatic fibrosis by inhibition of activated hepatic stellate and kupffer cells and by reducing oxidative stress in the al group . additionally , ang - ii synthesis by cardiac fibroblasts , extracellular as well as intracellular , is catalyzed by ace . thus , ace inhibitors can prevent ang - ii synthesis by cardiac fibroblasts , and ang - ii receptor inhibitors can block autocrine / paracrine effects of extracellular ang - ii . another study has been shown that inhibition of locally produced ang - ii could decrease inflammation and fibrosis in kidney , lung , and liver tissues . because ace and at-1 plays a key role in liver fibrosis , ras was reported to be highly expressed in activated human hepatic stellate cells . moreover , an at-1 antagonist , losartan , also reduces the extent of liver damage . interference of ang - ii in adriamycin hepatotoxicity is associated with stimulating ros production in hepatocytes , mainly via at-1 and mitochondria - derived pathway because losartan can inhibit ang - ii - stimulated ros generation . these results are in line with our findings in the present study that attenuation of ang - ii production in the liver can modulate ros production , arising from attenuation of mitochondrial dysfunction in liver exposed to adr . according to our current results , ang - ii cooperates with adr to produce more oxidant , causing its hepatotoxicity ( figure 2 ) but not attenuation of antioxidants ( figure 3 ) . the first is that crosstalk between adr and ang - ii might be based more on oxidant production than changing of antioxidant . most hepatic damage by ang - ii is known to be related to elevated oxidants . since the liver plays an important role in pro - oxidants , detoxification is known to have the highest content of antioxidants , a decline antioxidant levels in the liver tissue may need enormous oxidant production to significant alternation of oxidant . thirdly , our study investigated acute hepatotoxicity induced by adr and determined that time is an important factor in adr s cytotoxicity . more time may be needed to change the highest content of antioxidants in liver tissue , consistant with our results that antioxidant level was not significantly decreased in any experimental groups . moreover , the role of the ras in this experimental model suggests that intrahepatic ras is markedly upregulated , and ras inhibition attenuates adr s hepatotoxicity . moreover , it is possible to have an interaction between the systemic and intrahepatic ras . therefore , the present study used ace and renin inhibitors to block ras , especially local ones . since insufficient work has been done on mitochondria to clarify this issue , the full of molecular mechanisms underlying the link between adr and ang - ii are still unclear . however , angii has been shown to alter mmp   and to stimulate ros production . this overproduction of mitochondrial ros by ang - ii has been implicated in renal toxicity of adr   and also neurodegenerative diseases , ischemia - reperfusion injury , atherosclerosis , and aging . therefore , if ang - ii production could be inhibited , the mitochondrial dysfunction might be restored . the rising evidence of the beneficial effect of ang - ii inhibition suggests that a parallel intra - mitochondrial ras might be capable of angii synthesis to modulate ros activity . because of more exposure to a high rate of oxidative damage , mitochondria must be destroyed and replaced . a previous study suggested that mitochondria might have their functional ras , leading to the intra - mitochondrial generation of an - ii . it has been shown to alter mitochondrial number   or traffic to the surface of rodent mitochondria as well . an intracellular ras system might have a role as a local amplifier of ras signaling for attenuating a certain amount of systemic ang - ii . ang - ii has been suggested to have some role in development of mitochondrial failure , resulting in overloading ca , depolarization of mmp , and reducing atp production   in liver   and kidney exposed to adriamycin toxicity . the present study provides a novel mechanism of adr hepatotoxicity in which adr might crosstalk with local ras , especially mitochondria , in the rat the liver . therefore , our current research strongly suggests that protecting against adr s adverse effect on liver tissue can pharmacologically antagonize local angiotensin - ii pathological effects , including oxidative stress , decreasing mitochondrial membrane potential and atp production . further studies are necessary to test the beneficial effect of ang - ii inhibition on adr s hepatotoxicity and the other cytotoxicity in animal and human studies .