in 2010 an estimated 142,570 people developed colorectal cancer ( crc ) with an estimated 51,370 people dying of the disease . synchronous liver metastases are found in 20% of patients , and more than half of those diagnosed with crc will go on to develop metachronous liver metastases [ 2 , 3 ] . liver only or liver - predominant disease affects 2035% of patients , affording those with resectable lesions the possibility of long - term survival . in selected cases with r0 resection , 10-year overall survival has been reported in the literature to range from 1725% [ 4 , 5 ] . in addition to its adjuvant use in stage 3 colon cancer and following hepatic resection , chemotherapy has the potential to convert borderline or unresectable liver disease to resectable disease by reducing the size of the tumor to an amenable dimension . furthermore , neoadjuvant chemotherapy has been advocated as a test for aggressive tumor biology [ 68 ] . timing and appropriateness of chemotherapy , however , is debated , and there are concerns regarding worse outcomes in heavily treated patients . in this regard , steatohepatitis , steatosis , and sinusoidal injury have been linked to the use of irinotecan , fluoropyrimidines and oxaliplatin . these models have yet to be applied to the study of commonly used agents for crc . given first - line use of oxaliplatin and irinotecan for stage iv crc , these agents were chosen for investigation . we hypothesized that posthepatectomy liver regeneration is impaired by oxaliplatin and/or irinotecan administration and that this impairment can be demonstrated in a mouse   model . eight - week - old c57bl/6 male mice , weighing between 2325 grams , were obtained from commercial sources ( taconic farms , hudson , ny ) . the animals were housed under standard 12-hour light/12-hour dark conditions with standard feed and water ad libitum . after a minimum of 48 hours acclimation , animals were randomized to receive either oxaliplatin ( 15  mg / kg ) , irinotecan ( 100 mg / kg or 250 mg / kg ) or control solution ( dextrose 5% water ) by intraperitoneal injection . animal handling , drug administration , monitoring , and survival surgery protocols were approved by the city of hope , research animal care committee . oxaliplatin and irinotecan were obtained through the city of hope , investigational drug services and diluted in non - chloride - containing solution ( dextrose 5% water ) to deliver the determined dose in an approximate volume of 100  mcl . dose regimens were based on data from in vivo activity in previously described colon cancer tumor models in mice [ 12 , 13 ] . irinotecan was administered at two dose levels as follows : regimen a , 100  mg / kg , ip divided in 2 weekly doses and regimen b , 250  mg / kg ip divided in 3 weekly doses ( 75  mg / kg , 75  mg / kg , 100  mg / kg ) . fourteen days after the last control or chemotherapy injection , a 70% hepatectomy was performed . despite using well - established dosing schedule   in a dedicated vivarium with skilled personnel , the left and median lobes were resected with preservation of the gallbladder for 70% hepatectomy . briefly , tribromoethanol ( avertin ) anesthetic was administered ip ( 250  mg / kg ) . after sterile prep a subxiphoid transverse incision was created and the median and left liver lobes were exteriorized . the lobes were encircled with silk ligature , their vascular pedicles tied at the base and the lobes resected . buprenorphine was administered ( 0.5  mg / kg subcutaneously ) upon awakening . at postoperative days 0 , 1 , 2 , 3 , and 7 , remnant right and caudate lobes were harvested , and blood was collected from the retroorbital sinus concomitant with animal sacrifice . in the oxaliplatin experimented cohort , percent liver regrowth was calculated by the following formula : ( mass of regenerating liver remnant in grams )  ( mass of resected liver lobes in grams)/(0.7 )  100 . in vivo brdu staining was accomplished by intraperitoneal injection of brdu ( 100  mg / kg ) 2 hours prior to sacrifice . uniform samples of hepatic parenchyma were removed and fixed in 4% formaldehyde solution , embedded in paraffin , sectioned at 5 micrometers , and stained with hematoxylin and eosin . the number of positively stained nuclei was counted in 3 randomly selected high - power fields per sample , one sample from at least 2  mice per time point and arm . under anesthesia prior to sacrifice , approximately 500  mcl of blood was drawn from the retro - orbital sinus and placed in serum separator tubes ( falcon ) . collected serum was then analyzed for alt after 10-fold dilution in 7% bovine serum albumin . 22 animals underwent 70% hepatectomy in the oxaliplatin versus control study , 9 animals in the control arm , and 13 in the oxaliplatin arm . animal weights of the survivors were similar to those of the control group at the time of hepatectomy . there were 3 perioperative deaths ; 1 in the control arm and 2 in the oxaliplatin arm which were technical in nature ( pneumothorax , excessive manipulation of lobes on extraction , and hemorrhage ) . percent liver regrowth ( table 1 and figure 1 ) at day 1 following hepatectomy did not differ between oxaliplatin - treated and control mice ( 56.1% versus 52.5% , resp . data collected on day 2 suggests less regrowth in the oxaliplatin - treated arm ( 57.6% versus 73.0% , p = 0.154 ) ; however this was not statistically significant . regeneration was significantly less in the treatment arm at day 3 ( 70.0% versus 89.0% , p = 0.048 ) . by 7 days following hepatectomy , delayed lr in the oxaliplatin - treated arm was no longer found to be statically significant ( 89.8% versus 99.0% , p = 0.214 ) . alt levels in oxaliplatin - treated animals were not found to be statistically different than controls throughout the study ( figure 5 ) . brdu incorporation was used to determine if oxaliplatin impairs dna synthesis ( cellular division ) , thus contributing to impaired liver regrowth ( figure 3 ) . oxaliplatin - treated animals showed significantly less incorporation consistent with reduced dna synthesis . in the irinotecan experiments no animals experienced chemotherapy - related mortality . weights were similar between groups at the time of hepatectomy . neither dose level , group a ( 100  mg / kg , n = 15 ; control n = 8) nor group b ( 250  mg / kg , n = 17 ; control n = 5 ) showed significant impairment in liver regrowth by mass compared with respective controls ( figure 2 ) . similar to the oxaliplatin group , irinotecan - treated animals showed peak alt levels at day 1 with return to baseline between days 37 ( data not shown ) . in contrast to the oxaliplatin results , brdu incorporation in irinotecan - treated animals was similar or increased compared to controls ( figure 4 ) . histologic examination of regenerating liver specimens showed no evidence of hepatic sinusoidal obstruction in oxaliplatin - or - irinotecan treated animals . mild ballooning changes due to increased cytoplasmic water were seen in both treated and untreated groups . in the oxaliplatin arm , mild portal inflammation with necrosis near the portal triads and microvesicular steatosis were seen in two animals , one at posthepatectomy day 2 and one at day 3 . the liver 's remarkable ability to restore a functionally adequate portion of its previous volume following surgical resection is tightly regulated by mechanisms that include bile acid interactions with the fxr nuclear receptor and several other complex mechanisms [ 11 , 14 ] . the mouse liver regeneration model is well described and highly reproducible in this posthepatectomy setting . the differences in regeneration are demonstrated at early timepoints , namely , days 2 and 3 after hepatectomy . we chose to apply this model to the study of liver regeneration after treatment with commonly used modern chemotherapeutic agents for crc . the oxaliplatin dose was selected based on established , species - specific doses from the research literature . an unexpected toxicity ( mortality ) the animal deaths affected the group sizes , but only impacted the planned experimental animal numbers ( approved by the research animal care committee ) that would be required to achieve definitive statistical results in one cohort . we discovered that oxaliplatin - treated animals showed significantly reduced regrowth on the third posthepatectomy day . this finding has not been previously described in a preclinical model and may , in part , be due to the mechanism of oxaliplatin cytotoxicity . oxaliplatin is a third - generation platinum derivative that acts at the level of dna by forming bulky dna adducts . most commonly , intrastrand links between guanine and adenine are formed by the platinum moiety . dna synthesis is impaired by these adducts which in turn leads to strand breaks and subsequent apoptosis . oxaliplatin 's mechanism of action is consistent with the marked decrease in dna synthesis demonstrated by the decrease in brdu staining in these experiments . analysis of oxaliplatin 's impact on brdu staining were demonstrated with significant differences as early as day 1 following hepatectomy with significant impairment in dna synthesis continuing through day 3 . these data combined with the absence of direct hepatic damage , as evidenced by nonsignificant differences in alt , suggest oxaliplatin blunts lr by inhibiting cell division in the early postoperative period . however , this effect appears to be lost by 7 days postoperatively as physiologic mechanisms to restore appropriate liver function normalize liver size by this time . despite the consistency ( growth and dna synthetic activity ) of these data , the small number of experimental animals requires they be viewed as exploratory , not definitive . the experimental model is well established , and the brdu incorporation is a sensitive measure of dna synthesis . the current literature contains variable conclusions on the impact of chemotherapy on liver regeneration . in part , this is due to differences in experimental modeling ( e.g. , number of chemotherapy injections , use of ki-67 , and single time - point analysis ) . the current series of experiments provides sequential time point evaluation at 0 , 1 , 2 , 3 , and 7 days in an attempt to mimic the immediate , early , and longer phases of hepatic regeneration in the human . this topic area remains controversial , and additional experiments with a consistent experimental model will be the most definitive way to answer the controversies and variability in results . irinotecan - treated animals did not show differences in liver regrowth despite treatment at previously documented pharmacologically active doses [ 16 , 17 ] . brdu incorporation assays corroborate these findings with no decrease but rather , a nonstatistically significant increase in dna synthesis . increased dna synthesis with irinotecan treatment may be related to the drug 's mechanism of action . irinotecan inhibits topoisomerase i , stabilizes single - strand breaks and results in double - strand breakage though interaction at the replication fork . our results suggest that the structure and coiling of dna is altered by irinotecan without direct effect on the cells ability to synthesize dna and thus incorporate brdu . despite the somewhat counterintuitive nature of this finding , a higher proportion of cells in s - phase after irinotecan treatment has been described in animal and clinical settings . this occurred despite the use of previously reported doses . in our experiments , although the toxicity was high during the administration of oxaliplatin , the surviving animals were fully recovered prior to hepatectomy with no difference in animal weight or appearance in the oxaliplatin - treated animals when compared to irinotecan - treated animals . this argues that differences in regrowth were liver specific and not a byproduct of other factors such as a weakened state or poor nutrition . recently , clinical studies have raised concerns regarding the significant hepatotoxicity of chemotherapeutic agents for crc . syndrome from hepatic sinusoidal obstruction , and worse posthepatectomy outcome is reported in association with chemotherapy - related steatohepatitis primarily with irinotecan [ 22 , 23 ] . histologic examination of the liver in 2 animals showed microvesicular steatosis and mild periportal inflammation . the differences seen in regeneration and dna synthesis , therefore , likely reflect changes not yet evident on h&e , but in part detectable with special staining techniques such as brdu immunohistochemistry . clinical guidelines for hepatectomy recommend more conservative volumes of liver resection in chemotherapy - treated patients with a goal future liver remnant of 30% , rather than 20% [ 16 , 24 ] . given the adverse effects of chemotherapy on the liver , our goal was to establish an animal model to study these interactions . these data suggest that early in the patient 's postoperative course , when the risk for liver failure is higher , regenerative mechanisms may be impaired . the mouse 70% hepatectomy model provides a useful tool for studying the effects of chemotherapy on posthepatectomy liver regeneration . we demonstrate that oxaliplatin impairs early liver regeneration in a posthepatectomy model and that this reduced regrowth correlates with decreased dna synthesis .
introduction . 
 we examined the murine hepatectomy model of liver regeneration ( lr ) in the setting of neoadjuvant chemotherapy . 
 methods . 
 c57bl/6 mice were randomized to receive neoadjuvant intraperitoneal ( ip ) injections of a control , oxaliplatin ( 15  mg / kg ) , or irinotecan ( 100  mg / kg or 250  mg / kg ) solution . 
 hepatectomy ( 70% ) was performed 14 days after the final ip treatment . 
 animals were sacrificed at postoperative day ( d ) 0 , 1 , 2 , 3 , and 7 . 
 liver remnants and serum were collected for analysis . 
 t - tests for independent samples were used for statistical comparisons . 
 results . 
 for oxaliplatin , percent lr did not differ at d1 or d2 but was significantly less at d3 ( 89.0% versus 70.0% , p = 0.048 ) with no difference on d7 ( p = 0.21 ) . 
 irinotecan - treated mice at both dose levels ( 100  mg / kg and 250  mg / kg ) showed no significant differences in lr . 
 brdu incorporation was significantly decreased in oxaliplatin - treated animals ( d1,2,3 ) . 
 conclusions . 
 neoadjuvant oxaliplatin but not irinotecan impairs early lr in a posthepatectomy murine model which correlates with decreased dna synthesis .