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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.
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