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Bhatia et al. Hepatoma Res 2018;4:9 I http://dx.doi.org/10.20517/2394-5079.2018.04 Page 3 of 16

synergistic effects of its multiple phytochemicals [19,20] . The ameliorative potential of lycopene enriched
extract has been found in patients of oesophageal cancer . The consistently reduced risk of chronic diseases
[21]
associated with increased consumption of lycopene enriched products provides a strong foundation for its
use as a potent chemopreventive agent against liver cancer.

Our earlier studies have reported the delay in progression of hepatic cancer upon lycopene enriched tomato
extract (LycT) consumption which was revealed by reduced histopathological alterations, improved survival
rate, reduced tumor incidence and burden [3,18] . This was also evident through modulation in the expression
of apoptosis and cell proliferation associated genes which further interferes in the progression of tumor
cells [18,22] . We have also found that LycT consumption aided in up regulating the detoxification system,
reducing chromosomal aberrations and modulating physiochemical characteristics of hepatocellular
membrane . Recently, in our laboratory, the role of LycT in inhibiting multiple dysregulated pathways
[12]
including hypoxia, angiogenesis and metastasis has also been delineated. The study suggested that it does
so by attenuating the expression of hypoxia inducible factor-alpha, vascular endothelial growth factor,
[23]
cluster of differentiation 31, matrix metalloproteinases (MMP)-2 and MMP-9 . Moreover, the modulation
of hepatic tumor marker [alpha fetoprotein (AFP)] and hepatic functional markers by LycT was also
[23]
demonstrated .
Thus the current scientific scenario has prompted us to study HCC during its early stages of development by
analyzing a panel of hematological, inflammatory and blood antioxidant markers whose dysfunction may be
related to critical events in hepatic cancer progression and their intervention with LycT. The assessment of
these markers in blood on a regular basis along with AFP and liver function markers may allow earlier HCC
detection. In addition, the physiological perturbations occurring in the hepatic tissue during carcinogenesis
was also assessed using Tc-mebrofenin hepatobiliary functional test.
99m

METHODS
Animal model for development of HCC
Female Balb/c mice (25-30 g) procured from the Central Animal House facility of Panjab University,
Chandigarh (India) were provided standard animal pellet diet (Ashirwad Industries, Kharar, Punjab,
India) and drinking water ad libitum. The animal house was maintained at a controlled temperature of
21 ºC ± 1 ºC and humidity of 50%-60% with a 12-h dark and light cycle. All the experimental studies
were performed in accordance with the Indian National Science Academy Guidelines for the use and
care of experimental animals and were initially approved by the Institutional Animal Ethics Committee
(IAEC), Panjab University, Chandigarh (IAEC/284-295 at Sr. No. 47). The mice were acclimatized to the
experimental conditions for duration of 1 week prior to the commencement of various treatments. LycT
was extracted from red tomatoes using hexane/acetone/ethanol as an extraction medium as described by
Gupta et al. . The content of lycopene in the extract was estimated using UV-VIS spectrophotometer as
[3]
described earlier . LycT in the upper hexane layer showed the presence of three characteristic peaks, i.e.,
[3]
at 444, 470 and 503 nm. Lycopene quantification was performed at 503 nm as to avoid the interferences
from other carotenoids including β-carotene, lutein, neoxanthin, etc. [24,25] . The average lycopene content
was approximately 14 mg/kg tomato .
[3]
Female Balb/c mice were randomly segregated into 4 groups. Animals of group 1 (control) were given vehicle (olive oil)
treatment orally thrice a week. Animals of group 2 (NDEA) and group 4 (LycT + NDEA) received an intraperitoneal
injection of NDEA at a cumulative dose rate of 200 mg/kg body weight for a total duration of 8 weeks. Group 3 (LycT)
and group 4 animals were administered LycT in olive oil orally at a dose rate of 5 mg/kg body weight thrice a week for
10 weeks. Oral administration of LycT was commenced 2 weeks prior to NDEA treatment and continued until
the termination of experimental period in LycT + NDEA group.