Oxaliplatin but not irinotecan impairs posthepatectomy liver regeneration in a murine model.

Abstract

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.

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SAGE-Hindawi Access to Research
International Journal of Hepatology
Volume 2011, Article ID 490463, 6 pages
doi:10.4061/2011/490463
Research Article
Oxaliplatinbut NotIrinotecan ImpairsPosthepatectomy Liver
Regenerationina MurineModel
PerryA.Soriano,1Nian Liu,2ErickCastillo,1Brock Foster,1AvoArtinyan,1Joseph Kim,1
Wendong Huang,2andLawrenceD.Wagman3
1Liver Tumor Program, City of Hope, 1500 E. Duarte Road, Duarte, CA 91010, USA
2Center for Gene Regulation and Drug Discovery, Beckman Research Institute, City of Hope, 1500 E. Duarte Road, Duarte,
CA 91010, USA
3The Center for Cancer Prevention and Treatment, St. Joseph Hospital, 1000 West La Veta Avenue, Orange, CA 92868, USA
Correspondence should be addressed to Lawrence D. Wagman, lawrence.wagman@stjoe.org
Received 11 May 2011; Revised 1 September 2011; Accepted 7 September 2011
Academic Editor: Wolfram Trudo Knoefel
Copyright © 2011 Perry A. Soriano et al. This is an open access article distributed under the Creative Commons Attribution
License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly
cited.
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
(15mg/kg), or irinotecan (100mg/Kg or 250mg/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
miceatbothdoselevels(100mg/Kgand250mg/Kg)showednosignificantdifferencesinLR.BrdUincorporationwassignificantly
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.
1.Introduction
2010 an estimated 142,570 people developed colorectal
cancer (CRC) with an estimated 51,370 people dying of the
disease [1]. 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
20–35% 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 17–25% [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 [6–8].
Timing and appropriateness of chemotherapy, however, is
debated, and there are concerns regarding worse outcomes
in heavily treated patients [9]. In this regard, steatohepatitis,
steatosis, and sinusoidal injury have been linked to the use
of irinotecan, fluoropyrimidines and oxaliplatin [10].
Animal models for the study of posthepatectomy liver
regeneration are well described [11]. 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 hypoth-
esized that posthepatectomy liver regeneration is impaired
by oxaliplatin and/or irinotecan administration and that this
impairment can be demonstrated in a mouse model.
2.MaterialsandMethods
2.1. Animal Maintenance and Treatments. Eight-week-old
C57BL/6 male mice, weighing between 23–25 grams, were

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2International Journal of Hepatology
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
(15mg/Kg), irinotecan (100 mg/kg or 250 mg/kg) or control
solution (dextrose 5% water) by intraperitoneal injection.
Animal tolerance of chemotherapy was closely monitored,
and posthepatectomy animals were evaluated daily. Animal
handling, drug administration, monitoring, and survival
surgery protocols were approved by the City of Hope,
Research Animal Care Committee.
2.2. Chemotherapy. Oxaliplatin and irinotecan were ob-
tained through the City of Hope, Investigational Drug Ser-
vices and diluted in non-chloride-containing solution (dex-
trose 5% water) to deliver the determined dose in an ap-
proximate volume of 100mcL. Dose regimens were based
on data from in vivo activity in previously described colon
cancer tumor models in mice [12, 13]. Oxaliplatin 15mg/Kg
wasadministeredIP ×1dose.Irinotecanwasadministeredat
two dose levels as follows: regimen A, 100mg/Kg, IP divided
in 2 weekly doses and regimen B, 250mg/Kg IP divided in
3 weekly doses (75mg/Kg, 75mg/Kg, 100mg/Kg). Fourteen
days after the last control or chemotherapy injection, a 70%
hepatectomy was performed. Despite using well-established
dosing schedule [12] in a dedicated vivarium with skilled
personnel, 19 of 32 animals died from the initial treatment
with oxaliplatin. There was no mortality in the irinotecan
group. All surviving animals were included in the surgical
portion of the experiment.
2.3. Animal Surgery. The left and median lobes were resected
with preservation of the gallbladder for 70% hepatectomy.
Briefly, tribromoethanol (Avertin) anesthetic was adminis-
tered IP (250mg/Kg). After sterile prep a subxiphoid tran-
sverse 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. Care was taken to spare the gallbladder
and associated bile ducts. Closure was accomplished with
autoclips. Buprenorphine was administered (0.5mg/Kg sub-
cutaneously) upon awakening. At postoperative days 0,
1, 2, 3, and 7, remnant right and caudate lobes were har-
vested, and blood was collected from the retroorbital
sinus concomitant with animal sacrifice. In the oxaliplatin
experimented cohort, there were 3 perioperative deaths (2
oxaliplatin treated, 1 control). There was no mortality in the
irinotecan cohort.
2.4. Percent Liver Regeneration by Mass. Percent liver re-
growth was calculated by the following formula: (Mass of
regenerating liver remnant in grams) ÷ (Mass of resected
liver lobes in grams)/(0.7) × 100.
2.5. Liver Histology and Bromodeoxyuridine (BrdU) Incor-
poration. In vivo BrdU staining was accomplished by intra-
peritoneal injection of BrdU (100mg/Kg) 2 hours prior
0
20
40
60
80
100
120
1234567
(day)
Regrowth (original mass %)
Control
Oxaliplatin
∗
∗
∗P = 0.0485
Figure 1: Liver regeneration by percent regrowth, oxaliplatin
15mg/Kg versus control, differs significantly at day 3.
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. BrdU immunohistochemical stain-
ing was performed using a commercially available kit
(Roche). The number of positively stained nuclei was count-
ed in 3 randomly selected high-power fields per sample, one
sample from at least 2mice per time point and arm.
2.6. ALT Analysis. Under anesthesia prior to sacrifice,
approximately 500mcL 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.
2.7. Statistical Analysis. Statistical comparisons were per-
formed using t-tests for independent samples.
3.Results
3.1. Oxaliplatin. 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., P =
0.312). 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%

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International Journal of Hepatology3
Table 1: Liver regeneration after oxaliplatin treatment, percent regrowth by mass, days 1, 2, 3, and 7.
Day
Day 1
Day 2
Day 3
Day 7
Regrowth control
56.1% (N = 2)
73.0% (N = 2)
89.0% (N = 2)
99.0% (N = 2)
Regrowth oxaliplatin
52.5% (N = 3)
57.6% (N = 3)
70.0% (N = 3)
89.8% (N = 2)
Independent T-test
P = 0.312
P = 0.154
P = 0.048
P = 0.214
N, number of animals per group.
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).
Hepatocyte injury was assessed by measurement of ALT
levels. ALT levels peaked at posthepatectomy day 1 and
normalize by day 3. 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). DNA synthesis was
significantly higher in the control arm at all three measured
timepoints. Oxaliplatin-treated animals showed significantly
less incorporation consistent with reduced DNA synthesis.
3.2. Irinotecan. 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 (100mg/Kg, N = 15; control N = 8)
nor group B (250mg/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 3–7 (data
not shown). In contrast to the oxaliplatin results, BRDU
incorporation in irinotecan-treated animals was similar or
increased compared to controls (Figure 4).
3.3. Histology. Histologic examination of regenerating liver
specimensshowednoevidenceofhepaticsinusoidalobstruc-
tion in oxaliplatin-or-irinotecan treated animals. Mild bal-
looning 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.
4.Discussion
The liver’s remarkable ability to restore a functionally ade-
quateportionofitspreviousvolumefollowingsurgicalresec-
tion 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 [11]. 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 [12]. An unexpected toxicity
(mortality) was observed in this experiment. 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 [15]. 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. BrdU
staining was more sensitive than percent regrowth by weight.
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 sequential
reporting is unique in investigations of this type. This topic
area remains controversial, and additional experiments with

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4 International Journal of Hepatology
0
10
20
30
40
50
60
70
80
90
Regrowth (original mass %)
Control
Irinotecan
1234567
(day)
(a)
Control
Irinotecan
1234567
(day)
0
20
40
60
80
100
Regrowth (original mass %)
(b)
Figure 2: Liver regeneration by percent regrowth does not dif-
fer after irinotecan treatment at cumulative 100mg/Kg (a) and
250mg/Kg (b) dose levels.
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
assayscorroboratethesefindingswithnodecreasebutrather,
a nonstatistically significant increase in DNA synthesis.
Increased DNA synthesis with irinotecan treatment may be
relatedtothedrug’smechanismofaction.Irinotecaninhibits
topoisomerase I, stabilizes single-strand breaks and results in
double-strand breakage though interaction at the replication
fork [18]. Our results suggest that the structure and coiling
of DNA is altered by irinotecan without direct effect on the
0
10
20
30
40
50
60
70
80
90
100
123
(day)
Number
positive nuclei per field
BrdU
Control
Oxaliplatin
Figure 3: BrdU incorporation is significantly less in oxaliplatin-
(15mg/kg) treated animals at days 1, 2, and 3.
0
10
20
30
40
50
60
70
80
BrdU positive nuclei per field
123
(day)
Control
Irinotecan
Figure 4: BrdU incorporation is increased in irinotecan-
(100mg/kg) treated animals.
cells ability to synthesize DNA and thus incorporate BrdU.
Despitethesomewhatcounterintuitivenatureofthisfinding,
a higher proportion of cells in S-phase after irinotecan
treatment has been described in animal and clinical settings
[19].
High toxicity was seen in the animals receiving oxali-
platin. This occurred despite the use of previously reported
doses [20]. 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 ani-
mals. This argues that differences in regrowth were liver

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International Journal of Hepatology5
0
1000
2000
3000
4000
1
234567
ALT (U/L)
Control
Oxaliplatin 15mg/Kg
P = 0.4
Figure 5: Representative plot of ALT levels showing a peak at day 1
with normalization between day 3 and 7.
specific and not a byproduct of other factors such as a weak-
ened state or poor nutrition.
Recently, clinical studies have raised concerns regarding
the significant hepatotoxicity of chemotherapeutic agents
for CRC [21]. Oxaliplatin is implicated in the “blue
liver” 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. However, this was an uncommon finding.
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 ani-
mal model to study these interactions. We have shown early
impairment of regenerative ability in oxaliplatin-treated
animals. These findings are corroborated by decreased DNA
synthesis. These data suggest that early in the patient’s
postoperative course, when the risk for liver failure is higher,
regenerative mechanisms may be impaired. Future studies
with this model will aim at abrogating these effects.
5.Conclusion
The mouse 70% hepatectomy model provides a useful tool
forstudyingtheeffectsofchemotherapyonposthepatectomy
liver regeneration. We demonstrate that oxaliplatin impairs
early liver regeneration in a posthepatectomy model and
that this reduced regrowth correlates with decreased DNA
synthesis.Conversely,irinotecandidnotimpairregeneration
or DNA synthesis.
Acknowledgments
The authors wish to thank members of the Department of
Pathology at City of Hope, namely, Sofia Loera and her
team for expert performance of the BrdU IHC staining, and
Dr. Peiguo Chu, MD, for prompt and thorough histologic
review.
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