- Email: [email protected]
Schistosoma mansoni infection: Is it a risk factor for development of hepatocellular carcinoma?
Acta Tropica 128 (2013) 542–547
Contents lists available at ScienceDirect
Acta Tropica journal homepage: www.elsevier.com/locate/actatropica
Schistosoma mansoni infection: Is it a risk factor for development of hepatocellular carcinoma? Manar Mahmoud El-Tonsy a , Hesham Mohammed Hussein a , Thanaa El-Sayed Helal b , Rania Ayman Tawfik a,∗ , Khalid Mohamed Koriem c , Hend Mohamed Hussein a a
Department of Parasitology, Faculty of Medicine, Ain Shams University, Abbassia, Cairo, Egypt Department of Pathology, Faculty of Medicine, Ain Shams University, Abbassia, Cairo, Egypt c Department of Medical Physiology, Medical Research Division, National Research Center, Cairo, Egypt b
a r t i c l e
i n f o
Article history: Received 24 August 2011 Received in revised form 30 June 2013 Accepted 26 July 2013 Available online 6 August 2013 Keywords: Schistosoma mansoni Carcinogenicity Hepatocellular carcinoma Diethylnitrosamine Alpha fetoprotein Ferritin
a b s t r a c t The burden of hepatocellular carcinoma (HCC) in Egypt has been increasing with a doubling in the incidence rate in the past 10 years, which necessitates the investigation of the possible risk factors to its development. The present study aimed at investigating the role of Schistosoma mansoni infection as a risk factor for development of HCC. Five hundred parasite free mice were categorized into four groups: Group I (induction of carcinoma by diethylnitrosamine (DEN)), Group II (DEN + Infection), Group III (Infection) and Group IV (Control). Groups I and II were further subdivided into 4 subgroups according to the dose of DEN given. Serum samples from each group were examined for levels of tumor markers alpha fetoprotein (AFP) and ferritin by ELISA, then mice were sacrificed and subjected to histopathological examination of their livers. These were repeated every week till the end of the experiment. The results of the histopathological examination clarified the role of S. mansoni in enhancing and aggravating the carcinogenic effect of DEN; dysplastic changes appeared earlier, with a higher grade and with a smaller dose of DEN in Group II compared to Group I. Serum levels of tumor markers showed earlier statistically significant differences in Group II than in Group I when compared to Group IV. We conclude that S. mansoni accelerates hepatic dysplastic changes in the presence of other risk factors making cancer appear early and with a more aggressive nature, compared to the same risk in absence of schistosomiasis. © 2013 Elsevier B.V. All rights reserved.
1. Introduction Schistosomiasis remains one of the most prevalent parasitic infections in the world. It has been estimated that more than 207 million people are infected and 779 million people are at risk of infection (Steinmann et al., 2006). Recent advances in the fields of molecular biology, epidemiology and infectious diseases have led to significant revelations to clarify the relationship between cancer and infective agents (Khurana et al., 2005). Schistosoma haematobium (S. haematobium) and S. japonicum infections have been found to be strongly associated with bladder cancer and hepatocellular carcinomas (HCC), respectively (Khurana et al., 2005). The international agency for research on cancer (IARC) considers S. haematobium infection a definitive cause of urinary bladder cancer with an associated 5-fold risk (Pisani et al., 1997). The evidence supporting role of S. japonicum in cancer occurrence is weaker, although it has been associated with both liver and colorectal cancer (Ishii et al., 1994). Also, Vennervald and Polman (2009)
∗ Corresponding author. Tel.: +20 100 1220490. E-mail address: ranya [email protected] (R.A. Tawfik). 0001-706X/$ – see front matter © 2013 Elsevier B.V. All rights reserved. http://dx.doi.org/10.1016/j.actatropica.2013.07.024
stated that many case reports linked liver cancer, colorectal cancer, prostate cancer and giant follicular lymphomas with S. mansoni infection. Hepatocellular carcinoma (HCC) is one of the most common cancers world-wide; it accounts for more than 90.0% of all primary hepatic tumors. The burden of HCC has been increasing in Egypt with a doubling in the incidence rate in the past 10 years. The rising incidence of HCC has been associated with increased prevalence of HCV infection (El-Serag, 2002). Clinical studies in Egypt had shown that 70–90% of patients with chronic hepatitis, cirrhosis or HCC have co-infection of schistosomiasis and HCV (Strickland, 2006). It was suggested that the combination of chronic schistosomiasis caused by S. mansoni and hepatitis B virus (HBV) or hepatitis C virus (HCV) may cause a higher risk of HCC as a result of increased viral load in co-infected patients leading to higher inflammatory activity as well as more advanced disease state (Kamal et al., 2000). Schistosomiasis can also initiate or promote neoplasia due to initiation of a chronic granulomatous inflammatory reaction that may block the venules, increase cell turnover, and promote the production of reactive oxygen and nitrogen species (ROS and RNOS) which have the potential to damage DNA, proteins and cell membranes, and modulate enzyme activities and gene expression
M.M. El-Tonsy et al. / Acta Tropica 128 (2013) 542–547
which promote carcinogenesis (Ohshima and Bartsch, 1994). Moreover, there is a strong correlation between schistosomiasis and P53 mutation (Liu et al., 2002). Habib et al. (2006) have pointed out that schistosomiasis mansoni and exposure to aflatoxin B1 act synergistically to increase the incidence of P53 gene mutation that enhances the progression of HCC at an early age. The aim of this study was to investigate the role of S. mansoni infection as a risk factor for development of HCC. To achieve this, mice were subjected to induction of HCC by different doses of the carcinogenic substance diethylnitrosamine (DEN) with and without S. mansoni infection and compared to control groups. 2. Materials and methods This study was carried out on experimental animals and had been performed in the Medical Physiology Department, National Research Center, Cairo, Egypt and Parasitology Department, Faculty of Medicine, Ain Shams University, Cairo, Egypt. 2.1. Groups of the study Mice animal model had been established as follows: 500 parasite free male Swiss albino mice of average weight 20–25 g were used in the study. They were divided into 4 groups as follows: 2.1.1. Group I (induction of carcinoma by DEN) This group was represented by 200 mice that were subjected to induction of hepatocellular carcinoma by different doses of carcinogenic substance, diethylnitrosamine (DEN) (Buchmann et al., 1991). A pilot study was done to determine the dose of DEN that causes pathological changes but does not lead to the death of mice. Accordingly, the dose of DEN was adjusted to be as follows: 1, 3, 5, and 10 g per gram of body weight and given to subgroups a, b, c, and d respectively. 2.1.2. Group II (DEN induction + S. mansoni infection) This group was represented by 200 mice that were subcutaneously infected with 50 cercariae of S. mansoni and exposed to induction of HCC by the same doses of DEN as Group I, after establishment of schistosomiasis, 8 weeks post infection. 2.1.3. Group III (S. mansoni infection) This group was represented by fifty mice that were subcutaneously infected with 50 cercariae of S. mansoni.
543
2.2.2. Induction of HCC Mice in Groups I (DEN) and II (DEN + Infection) were given a single intraperitoneal injection of diethylnitrosamine (DEN) according to Buchmann et al. (1991). 2.2.3. Collection of sera and measurement of biomarkers Blood samples were collected from each mouse in all studied groups (Harlow and Lane, 1988). Detection of Alpha fetoprotein (AFP) was carried out according to Engall et al. (1980) and ferritin according to White et al. (1986) by ELISA technique. 2.2.4. Dissection of mice and preparation of slides Formalin-fixed paraffin embedded tissue sections stained with Hematoxylin and Eosin (H&E), were used. These sections were examined for small liver cell dysplasia (SCD), which is defined as small hepatocytes with decreased cytoplasm and increased nucleocytoplasmic ratio, hyperchromatic nuclei with slight nuclear pleomorphism, arranged in compact areas. It is considered the first step in the development of hepatocellular carcinoma (Watanabe et al., 1983). The density of SCD was semiquantitavely graded according to Lee et al. (1997) into: 0 (absent), 1 (few scattered foci), 2 (occasional foci) and 3 (multiple or widespread foci). On the other hand, the severity of SCD was classified into low grade (1) and high grade (2) (Thomas et al., 1992). High grade SCD is defined as increase in chromatin density, multiple mitotic figures, marked fibrosis, thick plate cells and proliferation of irregular shaped basophilic liver cell cords. A total score of dysplasia was obtained by summation of the density of dysplasia and the severity of dysplasia. This score varied from 0 to 5. 2.3. Ethical considerations Animal experimental studies and sacrifice procedures complied with ethical guidelines approved by the Ethical Committee of the Federal Legislation, the National Institutes of Health Guidelines in the USA and with the current laws and regulations of the Egyptian Ministry of Higher Education and Scientific Research. 2.4. Statistical analysis The data were presented as mean values with standard deviations. Student’s t-test was used to analyze the difference between groups. Groups I and II were compared as regards the score of dysplasia using Chi square test. Results with p values less than 0.05 were considered statistically significant. Results with p values less than 0.001 were considered statistically highly significant.
2.1.4. Group IV (healthy control) This group was represented by fifty non-infected healthy, parasite free mice as a control group.
3. Results
2.2. Methods
No histopathological changes were observed in subgroups a, b and c of Group I (DEN) all over the study period and all specimens showed normal hepatic architecture and cellular structure. Also, there were no histopathological changes in subgroup a of Group II (DEN + Infection) throughout the study (Table 1). With dose 1 g of DEN, all mice of subgroups Ia (DEN) and IIa (DEN + Infection) did not show SCD (grade 0). With dose 3 g of DEN, all mice in subgroup Ib (DEN) did not show SCD (100% grade 0) while in subgroup IIb (DEN + Infection), 32% of mice showed grade 4 SCD (Table 2 and Fig. 1C) and 68% showed grade 5 SCD (Table 2 and Fig. 1D) with a highly significant statistical difference between the 2 groups. With dose 5 g of DEN, there was as a highly significant statistical difference between Groups I (DEN) and II (DEN + Infection)
One week after induction of hepatocellular carcinoma in Groups I and II, and establishment of schistosomiasis in Group III, serum samples were examined for the levels of AFP and ferritin by ELISA from each group. Then mice were sacrificed and subjected to histopathological examination of their livers. These random samples were repeated every week till the end of the experiment. 2.2.1. Infection of mice with S. mansoni Mice in Groups II (DEN + Infection) and III (Infection) were infected with 50 cercariae of S. mansoni using the ring method of subcutaneous infection described by Smithers and Terry (1965).
3.1. Liver histopathological results
544
M.M. El-Tonsy et al. / Acta Tropica 128 (2013) 542–547
Fig. 1. (A) Group IV showing normal hepatic architecture and cellular structure (H&E 200×) (B) Group III showing typical schistosomal hepatic granuloma (H&E 400×). (C) Subgroup IIb showing small cell dysplasia grade 4 in all the cells with schistosomal granulomas in between in the 1st week post induction (H&E 200×). (D) Subgroup IIb showing small cell dysplasia grade 5 in the 3rd week post induction. White arrows: mitotic figures and binucleation. Black arrow: proliferation of irregular shaped basophilic liver cell cords and fibrosis (H&E 400×).
Table 1 Total score of small cell dysplasia (SCD) in Groups I (DEN) and II (DEN + Infection). Groups
Group I
Sub-groups
a
b
c
d
Group II a
b
c
d
Wk 1 PI Wk 2 PI Wk 3 PI Wk 4 PI Wk 5 PI Wk 6 PI
0 0 0 0 0 0
0 0 0 0 0 0
0 0 0 0 0 0
2 2 2 2 4 4
0 0 0 0 0 0
4 4 5 5 5 5
4 5 5 5 5 5
4 5 5 5 5 5
A total score of dysplasia was obtained by summation of the density and the severity of dysplasia. This score varied from grade 0–5. No dysplastic changes were observed in the hepatic parenchyma in specimens of Groups III and IV all over the study period. PI: Post induction.
as all mice in subgroup Ic (DEN) did not show SCD (100% grade
0) while in subgroup IIc (DEN + Infection) 16% of mice showed grade 4 SCD and 84% showed grade 5 SCD. With dose 10 g of DEN, there was a highly significant statistical difference between Groups I (DEN) and II (DEN + Infection) as 64% of mice in subgroup Id (DEN) showed grade 2 SCD and 36% showed grade 4 SCD while no mice showed grade 5 SCD. Meanwhile, 16% of mice in subgroup IId (DEN + Infection) showed grade 4 SCD and 84% showed grade 5 SCD (Table 2). All in all, comparison between Group I (DEN) and Group II (DEN + Infection) as regards the frequency of different grades of SCD with all doses of DEN showed that there was a highly significant statistical difference between the 2 groups as 75% of mice in Group I (DEN) did not show SCD (grade 0), 16% showed grade 2 SCD, 9% showed grade 4 SCD and no mice showed grade 5 SCD. While in Group II (DEN + Infection), 25% did not show SCD (grade 0), 16% showed grade 4 SCD and 59% showed grade 5 SCD (Table 2).
Table 2 Comparison between Group I (DEN) and Group II (DEN + Infection) as regards the frequency of grades of SCD. Grade
Group I N (%)
Group II N (%)
X2
p
Sig.
All doses of DEN
0 2 4 5
150 (75.00) 32 (16.00) 18 (9.00) 0 (0.00)
50 (25.00) 0 (0.00) 32 (16.00) 118 (59.00)
204
<0.001
HS
Dose 3 g (subgroup b)
0 4 5
50 (100.00) 0 (0.00) 0 (0.00)
0 (0.00) 16 (32.00) 34 (68.00)
100
<0.001
HS
Dose 5 g (subgroup c)
0 4 5
50 (100.00) 0 (0.00) 0 (0.00)
0 (0.00) 8 (16.00) 42 (84.00)
100
<0.001
HS
Dose 10 g (subgroup d)
2 4 5
32 (64.00) 18 (36.00) 0 (0.00)
0 (0.00) 8 (16.00) 42 (84.00)
<0.001
HS
With dose 1 g of DEN, all mice of subgroups Ia (DEN) and IIa (DEN + Infection) did not show SCD.
77.8
M.M. El-Tonsy et al. / Acta Tropica 128 (2013) 542–547 Table 3 Comparison between the frequency of grades of SCD among Group I (DEN) and Group II (DEN + Infection) throughout the study period. Grade
Weeks 1–3 (%)
Weeks 4–6 (%)
X2
p
Sig.
Group I
0 2 4
75 25 0
75 7.69 17.31
25.9
<0.001
HS
Group II
0 4 5
25 33.33 41.67
25 0 75
44.1
<0.001
HS
The frequency of different grades of SCD in Group I (DEN) throughout the 6 weeks of the study showed that 75% of mice did not show SCD (grade 0) throughout the study, while 25% of mice showed grade 2 SCD in the first 3 weeks of the study as compared to 7.69% in the last 3 weeks of the study. No mice in Group I (DEN) showed either grade 4 or 5 in the first 3 weeks of the study, while 17.31% of mice in Group I (DEN) showed grade 4 SCD in the last 3 weeks of the study with a highly significant statistical difference (Table 3). The frequency of different grades of SCD among Group II (DEN + Infection) showed that 25% of mice did not show any SCD (grade 0) throughout the 6 weeks of the study, while 33.33% of mice showed grade 4 SCD in the first 3 weeks of the study and no mice showed grade 4 SCD in the last 3 weeks of the study. Meanwhile, 41.67% of mice in Group II (DEN + Infection) showed grade 5 SCD in the first 3 weeks of the study, while 75% of mice showed grade 5 SCD in the last 3 weeks of the study with a highly significant statistical difference (Table 3). The histopathological examination of the liver of mice in Group III (Infection) showed the typical granulomas around Schistosoma eggs, plus a varying degree of mild to moderate fibrosis confined mainly to the portal area. However, no dysplastic changes appeared all over their survival period that had extended to 13 weeks (Fig. 1B). The histopathological picture of the liver of Group IV (Control) remained normal in relation to both architecture and cellular structure throughout the study period (Fig. 1A).
3.2. Serological results of biomarkers 3.2.1. Results of serum AFP In the present study, serum levels of AFP of Groups I (DEN), II (DEN + Infection) and III (Infection) were compared to Group IV (Control). The results of AFP in subgroups Ia and Ib showed, a statistically significant difference in the 5th week when compared to the control group, while this difference was observed one week earlier (in the 4th week) in the same subgroups of Group II (DEN + Infection) compared to the control group. In subgroups Ic and IIc, a statistically significant difference was detected in the 4th week when compared to the control group, but the differences were more significant in subgroup IIc (Table 4). In subgroup Id, a highly statistically significant difference was detected in the 4th week compared to the control group, while this difference was observed two weeks earlier (in the 2nd week) in subgroup IId (Table 4). Comparing AFP levels of Group III (Infection) to Group IV (Control), no significant difference was found between the two groups all over the study period. Comparing AFP serum level of Group II (DEN + Infection) to Group I (DEN), significant statistical differences between these two groups started to appear in the 4th week in subgroups a, b and c, and in the 2nd week in subgroup d (Table 4).
545
3.2.2. Results of serum ferritin In the present study, serum levels of ferritin of Groups I (DEN), II (DEN + Infection) and III (Infection) were compared to Group IV (Control). The results of serum ferritin in subgroups Ia and Ib showed a statistically significant difference on the 2nd week post induction when compared to the control group (Table 5). This difference was observed one week earlier (in the 1st week) in subgroup IIb when compared to the control group. In subgroups c and d of Group I (DEN) and Group II (DEN + Infection), a statistically significant difference was detected on the 1st week post induction when compared to Group IV (Control). Comparing serum ferritin levels of Group III (Infection) to Group IV (Control), no significant difference was found between the two groups all over the study period. Comparing serum ferritin levels of Group II (DEN + Infection) to Group I (DEN), significant statistical differences between these two groups started to appear in the 2nd week in subgroup d (Table 5).
4. Discussion The alarming increase in the incidence rate of HCC has increased the need of investigating the contribution of the emerging risk factors to its development (Anwar et al., 2008). It was suggested that the combination of chronic schistosomiasis caused by S. mansoni and hepatitis B or hepatitis C virus may result in a higher risk of HCC (Kamal et al., 2000). Yosry (2006) stated that the link between S. mansoni and HCC appears to be an indirect one. Patients infected with schistosomiasis have an increased risk of chronic infection with HBV and HCV after an episode of acute viral hepatitis. These patients are at a higher risk of earlier deterioration of liver function, the development of cirrhosis, and more rapid progression toward end-stage liver disease and even HCC. The investigation of an infectious origin of cancer has always been appealing, since an infectious origin of cancer implies that a cancer can be preventable. Furthermore, the role of infections in carcinogenesis has given scientists insights into carcinogenesis in general (Abdel-Rahim, 2001). However, the role of S.mansoni infection in the development of HCC has not yet been well evaluated. In the present study, we investigated the role of S.mansoni infection as a risk factor for development of HCC. Gomaa et al. (2009) had considered histopathological examination as the chief method for a sure and earlier diagnosis of HCC. In the present study, SCD grade 4 appeared as early as the 1st week post induction in subgroup b of Group II (DEN + Infection) (Table 1 and Fig. 1C) as compared to Group I (DEN) in which SCD grade 2 started to appear only in subgroup d (Table 1). Thus, SCD appeared earlier, with a higher grade and with a smaller dose of DEN in Group II (DEN + Infection) when compared to Group I (DEN). Small cell dysplasia was described by Crawford (1990) and he stated that it is the more likely precursor for malignancy, based on its morphological similarities to HCC in both light and electron microscopy and on observations made in animal model that studies hepatic carcinogenesis. Ganne-Carrie et al. (1996) have clearly shown that the presence of small cell dysplasia in a liver biopsy is associated with a high risk of development of HCC. Moreover, Le Bail et al. (1997) in another study on premalignant hepatic lesions concluded that, the presence of liver cell dysplasia in a liver biopsy from a patient with cirrhosis should be considered a major risk factor for the presence of HCC. In the present study, there was no progression of SCD beyond grade 4 in Group I (DEN). On the other hand, Group II (DEN + Infection) showed progression of SCD to grade 5 starting from the 3rd week post induction in subgroup b (Fig. 1D) and the 2nd week post induction in subgroups c and d. In the present study, serum levels of AFP and ferritin of Groups I (DEN), II (DEN + Infection) and III (Infection) were compared to
546
M.M. El-Tonsy et al. / Acta Tropica 128 (2013) 542–547
Table 4 Results of serum AFP of all studied groups. Wk1
Wk2
Wk3
Group I compared to Group IV 7.81 ± 0.67 Ia Ib 7.92 ± 0.71 Ic 7.94 ± 0.56 Id 8.49 ± 0.67 Group IV 7.65 ± 0.84
8.15 8.41 8.5 8.75 7.87
± ± ± ± ±
0.78 0.9 0.74 0.83 0.79
Group II compared to Group IV IIa 7.65 ± 0.84 IIb 7.98 ± 0.75 IIc 8.18 ± 0.85 IId 8.5 ± 0.86 Group IV 7.65 ± 0.84
7.87 8.52 8.56 9.85 7.87
± ± ± ± ±
Group I compared to Group II Ia 7.81 ± 0.67 IIa 7.89 ± 0.67 Ib 7.92 ± 0.71 IIb 7.98 ± 0.75 Ic 7.94 ± 0.56 IIc 8.18 ± 0.85 Id 8.49 ± 0.67 IId 8.5 ± 0.86
8.15 8.46 8.41 8.52 8.5 8.56 8.75 9.85
± ± ± ± ± ± ± ±
Wk4
Wk5
Wk6
8.64 8.86 9.04 9.18 8.38
± ± ± ± ±
0.86 0.82 0.86 0.83 0.81
9 ± 0.97 9.1 ± 0.98 10.16 ± 1.02* 11.62 ± 1** 8.41 ± 0.87
9.75 11.54 12.65 13.51 8.36
± ± ± ± ±
0.83* 1.06** 1.17** 1.16** 0.79
13.2 14.21 15.3 17.24 8.94
± ± ± ± ±
1.05** 1.18** 1.24** 1.2** 0.89
0.79 0.9 0.76 0.79** 0.79
8.38 8.94 9.15 10.36 8.38
± ± ± ± ±
0.81 0.87 0.84 0.92** 0.81
8.41 ± 0.87* 10.28 ± 0.92** 11.28 + 0.95** 12.46 ± 1.05** 8.41 ± 0.87
8.36 12.6 13.42 15.28 8.36
± ± ± ± ±
0.79** 1.1** 1.21** 1.17** 0.79
8.94 16.38 17.58 18.15 8.94
± ± ± ± ±
0.89** 1.26** 1.38** 1.29** 0.89
0.78 0.81 0.9 0.9 0.74 0.76 0.83 0.79*
8.64 8.85 8.86 8.94 9.04 9.15 9.18 10.36
± ± ± ± ± ± ± ±
0.86 0.9 0.82 0.87 0.86 0.84 0.83 0.92*
9 ± 0.97 9.92 ± 0.84* 9.1 ± 0.98 10.28 ± 0.92* 10.16 ± 1.02 11.28 + 0.95* 11.62 ± 1 12.46 ± 1.05
9.75 10.2 11.54 12.6 12.65 13.42 13.51 15.28
± ± ± ± ± ± ± ±
0.83 0.95 1.06 1.1* 1.17 1.21 1.16 1.17*
13.2 15.28 14.21 16.38 15.3 17.58 17.24 18.15
± ± ± ± ± ± ± ±
1.05 1.12* 1.18 1.26* 1.24 1.38* 1.2 1.29
Data were expressed as mean ± SD. There was no significant difference between Group III (Infection) and Group IV (Control) all over the study period. * Statistical significant difference P < 0.05. ** Highly statistical significant difference P < 0.001.
In the present work, the earlier higher significant increase in the values of serum AFP and ferritin in subgroups a, b of Group II (DEN + Infection) compared to their corresponding subgroups of Group I (DEN) was obvious and matches with the more aggressive dysplastic changes that appeared in Group II (DEN + Infection) compared to Group I (DEN). The normal histopathological picture of Group III (Infection) also matches with the serum levels of biomarkers of this group that did not show any significant difference when compared to the control group all over the study period. These results agree with Gameel et al. (2009) who pointed out the role of serum ferritin in increasing the diagnostic power of AFP in early detection of HCC. Thus, the experimental results of the present study had clarified the role of S. mansoni in enhancing and aggravating the
Group IV (Control). AFP is a fetal specific glycoprotein produced primarily by the fetal liver. Normally, its serum concentration falls rapidly after birth and its synthesis in adult life is decreased. It is present in high concentrations in the serum of many patients with primary HCC and testicular tumors. Serum AFP is the most widely used marker for HCC, and it is considered by many researchers as the most useful tumor marker in high risk patients (Zhou et al., 2006). However, its sensitivity and specificity are poor. The falsenegative or positive rates with AFP as a tumor marker has increased the need to search for serologic markers that are specifically associated with HCC (Pleguezuelo et al., 2010). Some studies have supported the view that the simultaneous measurement of AFP and ferritin in serum is useful for the early detection of HCC in high risk patients (Nakano et al., 2008). Table 5 Results of serum ferritin of all studied groups. Wk1
Wk2
Wk3
Wk4
Wk5
Wk6
Group I compared to Group IV Ia 61.6 ± 2.54 Ib 62.3 ± 2.43 Ic 63.4 ± 2.58* Id 64.9 ± 2.63* Group IV 60.5 ± 2.76
63.5 64 65 65 60.3
± ± ± ± ±
2.91* 2.69* 2.76* 2.98* 2.79
64.8 65.7 66.1 68.2 60.6
± ± ± ± ±
2.78* 2.78** 2.97** 2.75** 2.84
66 66.3 68.4 73.5 60.7
± ± ± ± ±
2.95** 2.96** 2.85** 2.94** 2.87
70 74.8 75.2 76.1 60
± ± ± ± ±
3.18** 3.05** 3.18** 3.11** 2.79
76.4 79 80.6 82 60.6
± ± ± ± ±
3.45** 3.28** 3.31** 3.27** 2.89
Group II compared to Group IV 62.5 ± 2.73 IIa IIb 63.2 ± 2.7* IIc 64.2 ± 2.8* IId 65.3 ± 2.76* Group IV 60.5 ± 2.76
64.1 65.8 66.5 68 60.3
± ± ± ± ±
2.69* 2.81** 2.76** 2.98** 2.79
66 66.5 67 69.1 60.6
± ± ± ± ±
2.87** 2.94** 3.08** 2.85** 2.84
68.5 69.2 71.5 76.4 60.7
± ± ± ± ±
2.94** 2.86** 2.76** 3.09** 2.87
72.3 75.2 78 80.9 60
± ± ± ± ±
3.09** 3.12** 3.14** 3.21** 2.79
77.1 81 82.5 84.6 60.6
± ± ± ± ±
3.36** 3.34** 3.29** 3.30** 2.89
Group I compared to Group II Ia 61.6 ± 62.5 ± IIa 62.3 ± Ib 63.2 ± IIb 63.4 ± Ic 64.2 ± IIc 64.9 ± Id 65.3 ± IId
63.5 64.1 64 65.8 65 66.5 65 68
± ± ± ± ± ± ± ±
2.91 2.69 2.69 2.81 2.76 2.76 2.98 2.98*
64.8 66 65.7 66.5 66.1 67 68.2 69.1
± ± ± ± ± ± ± ±
2.78 2.87 2.78 2.94 2.97 3.08 2.75 2.85
66 68.5 66.3 69.2 68.4 71.5 73.5 76.4
± ± ± ± ± ± ± ±
2.95 2.94 2.96 2.86 2.85 2.76 2.94 3.09*
70 72.3 74.8 75.2 75.2 78 76.1 80.9
± ± ± ± ± ± ± ±
3.18 3.09 3.05 3.12 3.18 3.14 3.11 3.21*
76.4 77.1 79 81 80.6 82.5 82 84.6
± ± ± ± ± ± ± ±
3.45 3.36 3.28 3.34 3.31 3.29 3.27 3.30
2.54 2.73 2.43 2.7 2.58 2.8 2.63 2.76
Data were expressed as mean ± SD. There was no significant difference between Group III (Infection) and Group IV (Control) all over the study period. * Statistical significant difference P < 0.05. ** Highly statistical significant difference P < 0.001.
M.M. El-Tonsy et al. / Acta Tropica 128 (2013) 542–547
carcinogenic effect of DEN since dysplastic changes appeared with a more aggressive nature, and with a smaller dose of DEN in Group II (DEN + Infection) compared to Group I (DEN). Also, serum levels of different biomarkers showed earlier significant statistical difference in Group II (DEN + Infection) than in Group I (DEN) when compared to the Group IV (Control). Meanwhile, S. mansoni infection failed to initiate any dysplastic changes and the serum level of different biomarkers of Group III (Infection) did not show any significant difference compared to the control group throughout the study period. These experimental results agree with Ishii et al. (1994) who found that hepatocellular carcinoma appeared early and in large numbers of mice infected with S. japonicum and given a known carcinogen, compared to mice given the carcinogen only. He concluded that there might be a cause–result relationship between schistosomiasis japonicum and hepatocellular carcinoma. The association of S. mansoni infection and hepatocellular carcinoma was also recorded in a chimpanzee in West Africa. Hepatitis B virus and hepatitis C virus were excluded by serological test in that animal (Abe et al., 1993). From the present study, it is concluded that S. mansoni accelerates hepatic dysplastic changes in the presence of any other risk factor making the cancer appear early and with a more aggressive nature, compared to the same risk in absence of schistosomiasis. Therefore, patients with chronic schistosomal hepatosplenomegaly, especially those who acquired infection in childhood and whose schistosomiasis insult will have the chance to affect their livers for a longer period of time, must be followed up for fear of earlier development of HCC. References Abdel-Rahim, A.Y., 2001. Parasitic infections and hepatic neoplasia. Digestive Diseases 19, 288–291. Abe, K., Kagei, N., Teramura, Y., Ejima, H., 1993. Hepatocellular carcinoma associated with chronic Schistosoma mansoni infection in a chimpanzee. Journal of Medical Primatology 22, 237–239. Anwar, W.A., Khaled, H.M., Amra, H.A., El-Nezami, H., Loffredo, C.A., 2008. Changing pattern of hepatocellular carcinoma (HCC) and its risk factors in Egypt: possibilities for prevention. Mutation Research 659, 176–184. Buchmann, A., Bauer-Hofmann, R., Mahr, J., Norman, R., Schwarz, M., 1991. Mutational activation of c-Ha-ras gene in liver tumors of different rodent strains: correlation with susceptibility to hepatocarcinogenesis. Proceedings of the National Academy of Sciences 88, 911–915. Crawford, J.M., 1990. Pathologic assessment of liver cell dysplasia and benign liver tumors: differentiation from malignant tumors. Seminars in Diagnostic Pathology 7, 115–128. El-Serag, H.B., 2002. Hepatocellular carcinoma: an epidemiologic view. Journal of Clinical Gastroenterology 35, S72–S78. Engall, E., Van Vunakis, H., Langone, J.J., 1980. Methods in Enzymology, 70. Academic Press, New York, pp. 419–492. Gameel, M., El Assaly, N., Medany, H., El-Ashry, N., Mostafa, I., Abd El Raof, E., ElRasky, M., 2009. Evaluation of tumor markers panel in detection of HCC in HCV Egyptian patients. Research Journal of Medicine and Medical Sciences 4, 402–410.
547
Ganne-Carrie, N., Chastang, C., Chapel, E., Munz, C., Pateron, D., Sibony, M., Deny, P., Trinchet, J., Callard, P., Guettier, C., Beaugrand, M., 1996. Predictive score for the development of hepatocellular carcinoma and additional value of liver large cell dysplasia in western patients with cirrhosis. Hepatology 23, 1112–1118. Gomaa, A.I., Khan, S.A., Leen, E.L., Waked, I., Taylor-Robinson, S.D., 2009. Diagnosis of hepatocellular carcinoma. World Journal of Gastroenterology 15, 1301–1314. Habib, S.L., Said, B., Ahmed, T., Awad, M.D., Mostafa, H., Ronald, C., 2006. Novel adenine adducts, N7-guanine-AFB1 adducts, and p53 mutations in patients with schistosomiasis and aflatoxin exposure. Cancer Detection and Prevention 30, 491–498. Harlow, E., Lane, D., 1988. Sampling serum. In: Harlow, E., Lane, D. (Eds.), Antibodies: a Laboratory Manuals, 5, 1st ed. Cold Spring Harbor Laboratory, pp. 116–129. Ishii, A., Matsuoka, H., Aji, T., Ohta, N., Arimoto, S., Wataya, Y., Hayatsu, H., 1994. Parasitic infection and cancer: with special emphasis on Schistosoma japonicum infection (Trematoda). A review. Mutation Research 305, 273–281. Kamal, S., Madwar, M., Bianchi, L., Tawil, A.E., Fawzy, R., Peters, T., Rasen, J.W., 2000. Clinical virological and histopathological features: long-term follow-up in patients with chronic hepatitis C co-infected with S. mansoni. Liver 20, 281–289. Khurana, S., Dubey, M.L., Malla, N., 2005. Association of parasitic infections and cancers. Indian Journal of Medical Microbiology 23, 74–79. Le Bail, B., Bernard, P.H., Carles, J., Balabaud, C., Bioulac-Sage, P., 1997. Prevalence of liver cell dysplasia and association with HCC in a series of 100 cirrhotic liver explants. Journal of Hepatology 21, 835–842. Lee, R.G., Tsamandas, A.C., Demetris, A.J., 1997. Large cell change (liver cell dysplasia) and hepatocellular carcinoma in cirrhosis: matched case–control study, pathological analysis, and pathogenic hypothesis. Hepatology 26, 1415–1422. Liu, H., Wang, Y., Gui, S., 2002. The point mutation of p53 gene in HCC from Anhui Province a non HCC prevalence area in China. World Journal of Gastroenterology 3, 480–482. Nakano, S., Kumada, T., Sugiyama, K., Watahiki, H., Takeda, L., 2008. Clinical significance of serum ferritin determination for hepatocellular carcinoma. American Journal of Gastroenterology 79, 623–627. Ohshima, H., Bartsch, H., 1994. Chronic infections and inflammatory processes as cancer risk factors: possible role of nitric oxide in carcinogenesis. Mutation Research 305, 253–264. Pisani, P., Parkin, D.M., Munoz, N., Ferlay, J., 1997. Cancer and infection: estimates of the attributable fraction in 1990. Cancer Epidemiology, Biomarkers and Prevention 6, 387–400. Pleguezuelo, M., Lopez, L.M., Rodrigues, A., Montero, J.L., Briceno, J., Ciria, R., Muntane, J., de la Mata, M., 2010. Proteomic analysis for developing new biomarkers of hepatocellular carcinoma. World Journal of Hepatology 2, 127–135. Smithers, S.R., Terry, R.J., 1965. The infection of laboratory hosts with cercariae of Schistosoma mansoni and the recovery of adult worms. Parasitology 55, 695–700. Steinmann, P., Keiser, J., Bos, R., Tanner, M., Utzinger, J., 2006. Schistosomiasis and water resources development: systematic review meta-analysis, and estimates of people at risk. Lancet Infectious Diseases 6, 411–425. Strickland, G.T., 2006. Liver disease in Egypt: hepatitis C superseded schistosomiasis as a result of iatrogenic and biological factors. Hepatology 43, 915–922. Thomas, R.M., Berman, J.J., Yetter, R.A., Moore, G.W., Hutchins, G.M., 1992. Liver cell dysplasia: a DNA aneuploid lesion with distinct morphologic features. Human Pathology 23, 496–503. Vennervald, B.J., Polman, K., 2009. Helminths and malignancy. Parasite Immunology 31, 686–696. Watanabe, S., Okita, K., Harada, T., Kodama, T., Numa, Y., Takemoto, T., Takahashi, T., 1983. Morphologic studies of the liver cell dysplasia. Cancer 51, 2197–2205. White, D., Kramer, D., Johnson, G., Dick, F., Hamilton, H., 1986. Enzyme immunoassay (microtiter strips) for the quantitative determination of ferritin in human serum. American Journal of Clinical Pathology 72, 346–349. Yosry, A., 2006. Schistosomiasis and neoplasia. Contributions to Microbiology 13, 81–100. Zhou, L., Liu, J., Luo, F., 2006. Serum tumor markers for detection of hepatocellular carcinoma. World Journal of Gastroenterology 12, 1175–1181.
Contents lists available at ScienceDirect
Acta Tropica journal homepage: www.elsevier.com/locate/actatropica
Schistosoma mansoni infection: Is it a risk factor for development of hepatocellular carcinoma? Manar Mahmoud El-Tonsy a , Hesham Mohammed Hussein a , Thanaa El-Sayed Helal b , Rania Ayman Tawfik a,∗ , Khalid Mohamed Koriem c , Hend Mohamed Hussein a a
Department of Parasitology, Faculty of Medicine, Ain Shams University, Abbassia, Cairo, Egypt Department of Pathology, Faculty of Medicine, Ain Shams University, Abbassia, Cairo, Egypt c Department of Medical Physiology, Medical Research Division, National Research Center, Cairo, Egypt b
a r t i c l e
i n f o
Article history: Received 24 August 2011 Received in revised form 30 June 2013 Accepted 26 July 2013 Available online 6 August 2013 Keywords: Schistosoma mansoni Carcinogenicity Hepatocellular carcinoma Diethylnitrosamine Alpha fetoprotein Ferritin
a b s t r a c t The burden of hepatocellular carcinoma (HCC) in Egypt has been increasing with a doubling in the incidence rate in the past 10 years, which necessitates the investigation of the possible risk factors to its development. The present study aimed at investigating the role of Schistosoma mansoni infection as a risk factor for development of HCC. Five hundred parasite free mice were categorized into four groups: Group I (induction of carcinoma by diethylnitrosamine (DEN)), Group II (DEN + Infection), Group III (Infection) and Group IV (Control). Groups I and II were further subdivided into 4 subgroups according to the dose of DEN given. Serum samples from each group were examined for levels of tumor markers alpha fetoprotein (AFP) and ferritin by ELISA, then mice were sacrificed and subjected to histopathological examination of their livers. These were repeated every week till the end of the experiment. The results of the histopathological examination clarified the role of S. mansoni in enhancing and aggravating the carcinogenic effect of DEN; dysplastic changes appeared earlier, with a higher grade and with a smaller dose of DEN in Group II compared to Group I. Serum levels of tumor markers showed earlier statistically significant differences in Group II than in Group I when compared to Group IV. We conclude that S. mansoni accelerates hepatic dysplastic changes in the presence of other risk factors making cancer appear early and with a more aggressive nature, compared to the same risk in absence of schistosomiasis. © 2013 Elsevier B.V. All rights reserved.
1. Introduction Schistosomiasis remains one of the most prevalent parasitic infections in the world. It has been estimated that more than 207 million people are infected and 779 million people are at risk of infection (Steinmann et al., 2006). Recent advances in the fields of molecular biology, epidemiology and infectious diseases have led to significant revelations to clarify the relationship between cancer and infective agents (Khurana et al., 2005). Schistosoma haematobium (S. haematobium) and S. japonicum infections have been found to be strongly associated with bladder cancer and hepatocellular carcinomas (HCC), respectively (Khurana et al., 2005). The international agency for research on cancer (IARC) considers S. haematobium infection a definitive cause of urinary bladder cancer with an associated 5-fold risk (Pisani et al., 1997). The evidence supporting role of S. japonicum in cancer occurrence is weaker, although it has been associated with both liver and colorectal cancer (Ishii et al., 1994). Also, Vennervald and Polman (2009)
∗ Corresponding author. Tel.: +20 100 1220490. E-mail address: ranya [email protected] (R.A. Tawfik). 0001-706X/$ – see front matter © 2013 Elsevier B.V. All rights reserved. http://dx.doi.org/10.1016/j.actatropica.2013.07.024
stated that many case reports linked liver cancer, colorectal cancer, prostate cancer and giant follicular lymphomas with S. mansoni infection. Hepatocellular carcinoma (HCC) is one of the most common cancers world-wide; it accounts for more than 90.0% of all primary hepatic tumors. The burden of HCC has been increasing in Egypt with a doubling in the incidence rate in the past 10 years. The rising incidence of HCC has been associated with increased prevalence of HCV infection (El-Serag, 2002). Clinical studies in Egypt had shown that 70–90% of patients with chronic hepatitis, cirrhosis or HCC have co-infection of schistosomiasis and HCV (Strickland, 2006). It was suggested that the combination of chronic schistosomiasis caused by S. mansoni and hepatitis B virus (HBV) or hepatitis C virus (HCV) may cause a higher risk of HCC as a result of increased viral load in co-infected patients leading to higher inflammatory activity as well as more advanced disease state (Kamal et al., 2000). Schistosomiasis can also initiate or promote neoplasia due to initiation of a chronic granulomatous inflammatory reaction that may block the venules, increase cell turnover, and promote the production of reactive oxygen and nitrogen species (ROS and RNOS) which have the potential to damage DNA, proteins and cell membranes, and modulate enzyme activities and gene expression
M.M. El-Tonsy et al. / Acta Tropica 128 (2013) 542–547
which promote carcinogenesis (Ohshima and Bartsch, 1994). Moreover, there is a strong correlation between schistosomiasis and P53 mutation (Liu et al., 2002). Habib et al. (2006) have pointed out that schistosomiasis mansoni and exposure to aflatoxin B1 act synergistically to increase the incidence of P53 gene mutation that enhances the progression of HCC at an early age. The aim of this study was to investigate the role of S. mansoni infection as a risk factor for development of HCC. To achieve this, mice were subjected to induction of HCC by different doses of the carcinogenic substance diethylnitrosamine (DEN) with and without S. mansoni infection and compared to control groups. 2. Materials and methods This study was carried out on experimental animals and had been performed in the Medical Physiology Department, National Research Center, Cairo, Egypt and Parasitology Department, Faculty of Medicine, Ain Shams University, Cairo, Egypt. 2.1. Groups of the study Mice animal model had been established as follows: 500 parasite free male Swiss albino mice of average weight 20–25 g were used in the study. They were divided into 4 groups as follows: 2.1.1. Group I (induction of carcinoma by DEN) This group was represented by 200 mice that were subjected to induction of hepatocellular carcinoma by different doses of carcinogenic substance, diethylnitrosamine (DEN) (Buchmann et al., 1991). A pilot study was done to determine the dose of DEN that causes pathological changes but does not lead to the death of mice. Accordingly, the dose of DEN was adjusted to be as follows: 1, 3, 5, and 10 g per gram of body weight and given to subgroups a, b, c, and d respectively. 2.1.2. Group II (DEN induction + S. mansoni infection) This group was represented by 200 mice that were subcutaneously infected with 50 cercariae of S. mansoni and exposed to induction of HCC by the same doses of DEN as Group I, after establishment of schistosomiasis, 8 weeks post infection. 2.1.3. Group III (S. mansoni infection) This group was represented by fifty mice that were subcutaneously infected with 50 cercariae of S. mansoni.
543
2.2.2. Induction of HCC Mice in Groups I (DEN) and II (DEN + Infection) were given a single intraperitoneal injection of diethylnitrosamine (DEN) according to Buchmann et al. (1991). 2.2.3. Collection of sera and measurement of biomarkers Blood samples were collected from each mouse in all studied groups (Harlow and Lane, 1988). Detection of Alpha fetoprotein (AFP) was carried out according to Engall et al. (1980) and ferritin according to White et al. (1986) by ELISA technique. 2.2.4. Dissection of mice and preparation of slides Formalin-fixed paraffin embedded tissue sections stained with Hematoxylin and Eosin (H&E), were used. These sections were examined for small liver cell dysplasia (SCD), which is defined as small hepatocytes with decreased cytoplasm and increased nucleocytoplasmic ratio, hyperchromatic nuclei with slight nuclear pleomorphism, arranged in compact areas. It is considered the first step in the development of hepatocellular carcinoma (Watanabe et al., 1983). The density of SCD was semiquantitavely graded according to Lee et al. (1997) into: 0 (absent), 1 (few scattered foci), 2 (occasional foci) and 3 (multiple or widespread foci). On the other hand, the severity of SCD was classified into low grade (1) and high grade (2) (Thomas et al., 1992). High grade SCD is defined as increase in chromatin density, multiple mitotic figures, marked fibrosis, thick plate cells and proliferation of irregular shaped basophilic liver cell cords. A total score of dysplasia was obtained by summation of the density of dysplasia and the severity of dysplasia. This score varied from 0 to 5. 2.3. Ethical considerations Animal experimental studies and sacrifice procedures complied with ethical guidelines approved by the Ethical Committee of the Federal Legislation, the National Institutes of Health Guidelines in the USA and with the current laws and regulations of the Egyptian Ministry of Higher Education and Scientific Research. 2.4. Statistical analysis The data were presented as mean values with standard deviations. Student’s t-test was used to analyze the difference between groups. Groups I and II were compared as regards the score of dysplasia using Chi square test. Results with p values less than 0.05 were considered statistically significant. Results with p values less than 0.001 were considered statistically highly significant.
2.1.4. Group IV (healthy control) This group was represented by fifty non-infected healthy, parasite free mice as a control group.
3. Results
2.2. Methods
No histopathological changes were observed in subgroups a, b and c of Group I (DEN) all over the study period and all specimens showed normal hepatic architecture and cellular structure. Also, there were no histopathological changes in subgroup a of Group II (DEN + Infection) throughout the study (Table 1). With dose 1 g of DEN, all mice of subgroups Ia (DEN) and IIa (DEN + Infection) did not show SCD (grade 0). With dose 3 g of DEN, all mice in subgroup Ib (DEN) did not show SCD (100% grade 0) while in subgroup IIb (DEN + Infection), 32% of mice showed grade 4 SCD (Table 2 and Fig. 1C) and 68% showed grade 5 SCD (Table 2 and Fig. 1D) with a highly significant statistical difference between the 2 groups. With dose 5 g of DEN, there was as a highly significant statistical difference between Groups I (DEN) and II (DEN + Infection)
One week after induction of hepatocellular carcinoma in Groups I and II, and establishment of schistosomiasis in Group III, serum samples were examined for the levels of AFP and ferritin by ELISA from each group. Then mice were sacrificed and subjected to histopathological examination of their livers. These random samples were repeated every week till the end of the experiment. 2.2.1. Infection of mice with S. mansoni Mice in Groups II (DEN + Infection) and III (Infection) were infected with 50 cercariae of S. mansoni using the ring method of subcutaneous infection described by Smithers and Terry (1965).
3.1. Liver histopathological results
544
M.M. El-Tonsy et al. / Acta Tropica 128 (2013) 542–547
Fig. 1. (A) Group IV showing normal hepatic architecture and cellular structure (H&E 200×) (B) Group III showing typical schistosomal hepatic granuloma (H&E 400×). (C) Subgroup IIb showing small cell dysplasia grade 4 in all the cells with schistosomal granulomas in between in the 1st week post induction (H&E 200×). (D) Subgroup IIb showing small cell dysplasia grade 5 in the 3rd week post induction. White arrows: mitotic figures and binucleation. Black arrow: proliferation of irregular shaped basophilic liver cell cords and fibrosis (H&E 400×).
Table 1 Total score of small cell dysplasia (SCD) in Groups I (DEN) and II (DEN + Infection). Groups
Group I
Sub-groups
a
b
c
d
Group II a
b
c
d
Wk 1 PI Wk 2 PI Wk 3 PI Wk 4 PI Wk 5 PI Wk 6 PI
0 0 0 0 0 0
0 0 0 0 0 0
0 0 0 0 0 0
2 2 2 2 4 4
0 0 0 0 0 0
4 4 5 5 5 5
4 5 5 5 5 5
4 5 5 5 5 5
A total score of dysplasia was obtained by summation of the density and the severity of dysplasia. This score varied from grade 0–5. No dysplastic changes were observed in the hepatic parenchyma in specimens of Groups III and IV all over the study period. PI: Post induction.
as all mice in subgroup Ic (DEN) did not show SCD (100% grade
0) while in subgroup IIc (DEN + Infection) 16% of mice showed grade 4 SCD and 84% showed grade 5 SCD. With dose 10 g of DEN, there was a highly significant statistical difference between Groups I (DEN) and II (DEN + Infection) as 64% of mice in subgroup Id (DEN) showed grade 2 SCD and 36% showed grade 4 SCD while no mice showed grade 5 SCD. Meanwhile, 16% of mice in subgroup IId (DEN + Infection) showed grade 4 SCD and 84% showed grade 5 SCD (Table 2). All in all, comparison between Group I (DEN) and Group II (DEN + Infection) as regards the frequency of different grades of SCD with all doses of DEN showed that there was a highly significant statistical difference between the 2 groups as 75% of mice in Group I (DEN) did not show SCD (grade 0), 16% showed grade 2 SCD, 9% showed grade 4 SCD and no mice showed grade 5 SCD. While in Group II (DEN + Infection), 25% did not show SCD (grade 0), 16% showed grade 4 SCD and 59% showed grade 5 SCD (Table 2).
Table 2 Comparison between Group I (DEN) and Group II (DEN + Infection) as regards the frequency of grades of SCD. Grade
Group I N (%)
Group II N (%)
X2
p
Sig.
All doses of DEN
0 2 4 5
150 (75.00) 32 (16.00) 18 (9.00) 0 (0.00)
50 (25.00) 0 (0.00) 32 (16.00) 118 (59.00)
204
<0.001
HS
Dose 3 g (subgroup b)
0 4 5
50 (100.00) 0 (0.00) 0 (0.00)
0 (0.00) 16 (32.00) 34 (68.00)
100
<0.001
HS
Dose 5 g (subgroup c)
0 4 5
50 (100.00) 0 (0.00) 0 (0.00)
0 (0.00) 8 (16.00) 42 (84.00)
100
<0.001
HS
Dose 10 g (subgroup d)
2 4 5
32 (64.00) 18 (36.00) 0 (0.00)
0 (0.00) 8 (16.00) 42 (84.00)
<0.001
HS
With dose 1 g of DEN, all mice of subgroups Ia (DEN) and IIa (DEN + Infection) did not show SCD.
77.8
M.M. El-Tonsy et al. / Acta Tropica 128 (2013) 542–547 Table 3 Comparison between the frequency of grades of SCD among Group I (DEN) and Group II (DEN + Infection) throughout the study period. Grade
Weeks 1–3 (%)
Weeks 4–6 (%)
X2
p
Sig.
Group I
0 2 4
75 25 0
75 7.69 17.31
25.9
<0.001
HS
Group II
0 4 5
25 33.33 41.67
25 0 75
44.1
<0.001
HS
The frequency of different grades of SCD in Group I (DEN) throughout the 6 weeks of the study showed that 75% of mice did not show SCD (grade 0) throughout the study, while 25% of mice showed grade 2 SCD in the first 3 weeks of the study as compared to 7.69% in the last 3 weeks of the study. No mice in Group I (DEN) showed either grade 4 or 5 in the first 3 weeks of the study, while 17.31% of mice in Group I (DEN) showed grade 4 SCD in the last 3 weeks of the study with a highly significant statistical difference (Table 3). The frequency of different grades of SCD among Group II (DEN + Infection) showed that 25% of mice did not show any SCD (grade 0) throughout the 6 weeks of the study, while 33.33% of mice showed grade 4 SCD in the first 3 weeks of the study and no mice showed grade 4 SCD in the last 3 weeks of the study. Meanwhile, 41.67% of mice in Group II (DEN + Infection) showed grade 5 SCD in the first 3 weeks of the study, while 75% of mice showed grade 5 SCD in the last 3 weeks of the study with a highly significant statistical difference (Table 3). The histopathological examination of the liver of mice in Group III (Infection) showed the typical granulomas around Schistosoma eggs, plus a varying degree of mild to moderate fibrosis confined mainly to the portal area. However, no dysplastic changes appeared all over their survival period that had extended to 13 weeks (Fig. 1B). The histopathological picture of the liver of Group IV (Control) remained normal in relation to both architecture and cellular structure throughout the study period (Fig. 1A).
3.2. Serological results of biomarkers 3.2.1. Results of serum AFP In the present study, serum levels of AFP of Groups I (DEN), II (DEN + Infection) and III (Infection) were compared to Group IV (Control). The results of AFP in subgroups Ia and Ib showed, a statistically significant difference in the 5th week when compared to the control group, while this difference was observed one week earlier (in the 4th week) in the same subgroups of Group II (DEN + Infection) compared to the control group. In subgroups Ic and IIc, a statistically significant difference was detected in the 4th week when compared to the control group, but the differences were more significant in subgroup IIc (Table 4). In subgroup Id, a highly statistically significant difference was detected in the 4th week compared to the control group, while this difference was observed two weeks earlier (in the 2nd week) in subgroup IId (Table 4). Comparing AFP levels of Group III (Infection) to Group IV (Control), no significant difference was found between the two groups all over the study period. Comparing AFP serum level of Group II (DEN + Infection) to Group I (DEN), significant statistical differences between these two groups started to appear in the 4th week in subgroups a, b and c, and in the 2nd week in subgroup d (Table 4).
545
3.2.2. Results of serum ferritin In the present study, serum levels of ferritin of Groups I (DEN), II (DEN + Infection) and III (Infection) were compared to Group IV (Control). The results of serum ferritin in subgroups Ia and Ib showed a statistically significant difference on the 2nd week post induction when compared to the control group (Table 5). This difference was observed one week earlier (in the 1st week) in subgroup IIb when compared to the control group. In subgroups c and d of Group I (DEN) and Group II (DEN + Infection), a statistically significant difference was detected on the 1st week post induction when compared to Group IV (Control). Comparing serum ferritin levels of Group III (Infection) to Group IV (Control), no significant difference was found between the two groups all over the study period. Comparing serum ferritin levels of Group II (DEN + Infection) to Group I (DEN), significant statistical differences between these two groups started to appear in the 2nd week in subgroup d (Table 5).
4. Discussion The alarming increase in the incidence rate of HCC has increased the need of investigating the contribution of the emerging risk factors to its development (Anwar et al., 2008). It was suggested that the combination of chronic schistosomiasis caused by S. mansoni and hepatitis B or hepatitis C virus may result in a higher risk of HCC (Kamal et al., 2000). Yosry (2006) stated that the link between S. mansoni and HCC appears to be an indirect one. Patients infected with schistosomiasis have an increased risk of chronic infection with HBV and HCV after an episode of acute viral hepatitis. These patients are at a higher risk of earlier deterioration of liver function, the development of cirrhosis, and more rapid progression toward end-stage liver disease and even HCC. The investigation of an infectious origin of cancer has always been appealing, since an infectious origin of cancer implies that a cancer can be preventable. Furthermore, the role of infections in carcinogenesis has given scientists insights into carcinogenesis in general (Abdel-Rahim, 2001). However, the role of S.mansoni infection in the development of HCC has not yet been well evaluated. In the present study, we investigated the role of S.mansoni infection as a risk factor for development of HCC. Gomaa et al. (2009) had considered histopathological examination as the chief method for a sure and earlier diagnosis of HCC. In the present study, SCD grade 4 appeared as early as the 1st week post induction in subgroup b of Group II (DEN + Infection) (Table 1 and Fig. 1C) as compared to Group I (DEN) in which SCD grade 2 started to appear only in subgroup d (Table 1). Thus, SCD appeared earlier, with a higher grade and with a smaller dose of DEN in Group II (DEN + Infection) when compared to Group I (DEN). Small cell dysplasia was described by Crawford (1990) and he stated that it is the more likely precursor for malignancy, based on its morphological similarities to HCC in both light and electron microscopy and on observations made in animal model that studies hepatic carcinogenesis. Ganne-Carrie et al. (1996) have clearly shown that the presence of small cell dysplasia in a liver biopsy is associated with a high risk of development of HCC. Moreover, Le Bail et al. (1997) in another study on premalignant hepatic lesions concluded that, the presence of liver cell dysplasia in a liver biopsy from a patient with cirrhosis should be considered a major risk factor for the presence of HCC. In the present study, there was no progression of SCD beyond grade 4 in Group I (DEN). On the other hand, Group II (DEN + Infection) showed progression of SCD to grade 5 starting from the 3rd week post induction in subgroup b (Fig. 1D) and the 2nd week post induction in subgroups c and d. In the present study, serum levels of AFP and ferritin of Groups I (DEN), II (DEN + Infection) and III (Infection) were compared to
546
M.M. El-Tonsy et al. / Acta Tropica 128 (2013) 542–547
Table 4 Results of serum AFP of all studied groups. Wk1
Wk2
Wk3
Group I compared to Group IV 7.81 ± 0.67 Ia Ib 7.92 ± 0.71 Ic 7.94 ± 0.56 Id 8.49 ± 0.67 Group IV 7.65 ± 0.84
8.15 8.41 8.5 8.75 7.87
± ± ± ± ±
0.78 0.9 0.74 0.83 0.79
Group II compared to Group IV IIa 7.65 ± 0.84 IIb 7.98 ± 0.75 IIc 8.18 ± 0.85 IId 8.5 ± 0.86 Group IV 7.65 ± 0.84
7.87 8.52 8.56 9.85 7.87
± ± ± ± ±
Group I compared to Group II Ia 7.81 ± 0.67 IIa 7.89 ± 0.67 Ib 7.92 ± 0.71 IIb 7.98 ± 0.75 Ic 7.94 ± 0.56 IIc 8.18 ± 0.85 Id 8.49 ± 0.67 IId 8.5 ± 0.86
8.15 8.46 8.41 8.52 8.5 8.56 8.75 9.85
± ± ± ± ± ± ± ±
Wk4
Wk5
Wk6
8.64 8.86 9.04 9.18 8.38
± ± ± ± ±
0.86 0.82 0.86 0.83 0.81
9 ± 0.97 9.1 ± 0.98 10.16 ± 1.02* 11.62 ± 1** 8.41 ± 0.87
9.75 11.54 12.65 13.51 8.36
± ± ± ± ±
0.83* 1.06** 1.17** 1.16** 0.79
13.2 14.21 15.3 17.24 8.94
± ± ± ± ±
1.05** 1.18** 1.24** 1.2** 0.89
0.79 0.9 0.76 0.79** 0.79
8.38 8.94 9.15 10.36 8.38
± ± ± ± ±
0.81 0.87 0.84 0.92** 0.81
8.41 ± 0.87* 10.28 ± 0.92** 11.28 + 0.95** 12.46 ± 1.05** 8.41 ± 0.87
8.36 12.6 13.42 15.28 8.36
± ± ± ± ±
0.79** 1.1** 1.21** 1.17** 0.79
8.94 16.38 17.58 18.15 8.94
± ± ± ± ±
0.89** 1.26** 1.38** 1.29** 0.89
0.78 0.81 0.9 0.9 0.74 0.76 0.83 0.79*
8.64 8.85 8.86 8.94 9.04 9.15 9.18 10.36
± ± ± ± ± ± ± ±
0.86 0.9 0.82 0.87 0.86 0.84 0.83 0.92*
9 ± 0.97 9.92 ± 0.84* 9.1 ± 0.98 10.28 ± 0.92* 10.16 ± 1.02 11.28 + 0.95* 11.62 ± 1 12.46 ± 1.05
9.75 10.2 11.54 12.6 12.65 13.42 13.51 15.28
± ± ± ± ± ± ± ±
0.83 0.95 1.06 1.1* 1.17 1.21 1.16 1.17*
13.2 15.28 14.21 16.38 15.3 17.58 17.24 18.15
± ± ± ± ± ± ± ±
1.05 1.12* 1.18 1.26* 1.24 1.38* 1.2 1.29
Data were expressed as mean ± SD. There was no significant difference between Group III (Infection) and Group IV (Control) all over the study period. * Statistical significant difference P < 0.05. ** Highly statistical significant difference P < 0.001.
In the present work, the earlier higher significant increase in the values of serum AFP and ferritin in subgroups a, b of Group II (DEN + Infection) compared to their corresponding subgroups of Group I (DEN) was obvious and matches with the more aggressive dysplastic changes that appeared in Group II (DEN + Infection) compared to Group I (DEN). The normal histopathological picture of Group III (Infection) also matches with the serum levels of biomarkers of this group that did not show any significant difference when compared to the control group all over the study period. These results agree with Gameel et al. (2009) who pointed out the role of serum ferritin in increasing the diagnostic power of AFP in early detection of HCC. Thus, the experimental results of the present study had clarified the role of S. mansoni in enhancing and aggravating the
Group IV (Control). AFP is a fetal specific glycoprotein produced primarily by the fetal liver. Normally, its serum concentration falls rapidly after birth and its synthesis in adult life is decreased. It is present in high concentrations in the serum of many patients with primary HCC and testicular tumors. Serum AFP is the most widely used marker for HCC, and it is considered by many researchers as the most useful tumor marker in high risk patients (Zhou et al., 2006). However, its sensitivity and specificity are poor. The falsenegative or positive rates with AFP as a tumor marker has increased the need to search for serologic markers that are specifically associated with HCC (Pleguezuelo et al., 2010). Some studies have supported the view that the simultaneous measurement of AFP and ferritin in serum is useful for the early detection of HCC in high risk patients (Nakano et al., 2008). Table 5 Results of serum ferritin of all studied groups. Wk1
Wk2
Wk3
Wk4
Wk5
Wk6
Group I compared to Group IV Ia 61.6 ± 2.54 Ib 62.3 ± 2.43 Ic 63.4 ± 2.58* Id 64.9 ± 2.63* Group IV 60.5 ± 2.76
63.5 64 65 65 60.3
± ± ± ± ±
2.91* 2.69* 2.76* 2.98* 2.79
64.8 65.7 66.1 68.2 60.6
± ± ± ± ±
2.78* 2.78** 2.97** 2.75** 2.84
66 66.3 68.4 73.5 60.7
± ± ± ± ±
2.95** 2.96** 2.85** 2.94** 2.87
70 74.8 75.2 76.1 60
± ± ± ± ±
3.18** 3.05** 3.18** 3.11** 2.79
76.4 79 80.6 82 60.6
± ± ± ± ±
3.45** 3.28** 3.31** 3.27** 2.89
Group II compared to Group IV 62.5 ± 2.73 IIa IIb 63.2 ± 2.7* IIc 64.2 ± 2.8* IId 65.3 ± 2.76* Group IV 60.5 ± 2.76
64.1 65.8 66.5 68 60.3
± ± ± ± ±
2.69* 2.81** 2.76** 2.98** 2.79
66 66.5 67 69.1 60.6
± ± ± ± ±
2.87** 2.94** 3.08** 2.85** 2.84
68.5 69.2 71.5 76.4 60.7
± ± ± ± ±
2.94** 2.86** 2.76** 3.09** 2.87
72.3 75.2 78 80.9 60
± ± ± ± ±
3.09** 3.12** 3.14** 3.21** 2.79
77.1 81 82.5 84.6 60.6
± ± ± ± ±
3.36** 3.34** 3.29** 3.30** 2.89
Group I compared to Group II Ia 61.6 ± 62.5 ± IIa 62.3 ± Ib 63.2 ± IIb 63.4 ± Ic 64.2 ± IIc 64.9 ± Id 65.3 ± IId
63.5 64.1 64 65.8 65 66.5 65 68
± ± ± ± ± ± ± ±
2.91 2.69 2.69 2.81 2.76 2.76 2.98 2.98*
64.8 66 65.7 66.5 66.1 67 68.2 69.1
± ± ± ± ± ± ± ±
2.78 2.87 2.78 2.94 2.97 3.08 2.75 2.85
66 68.5 66.3 69.2 68.4 71.5 73.5 76.4
± ± ± ± ± ± ± ±
2.95 2.94 2.96 2.86 2.85 2.76 2.94 3.09*
70 72.3 74.8 75.2 75.2 78 76.1 80.9
± ± ± ± ± ± ± ±
3.18 3.09 3.05 3.12 3.18 3.14 3.11 3.21*
76.4 77.1 79 81 80.6 82.5 82 84.6
± ± ± ± ± ± ± ±
3.45 3.36 3.28 3.34 3.31 3.29 3.27 3.30
2.54 2.73 2.43 2.7 2.58 2.8 2.63 2.76
Data were expressed as mean ± SD. There was no significant difference between Group III (Infection) and Group IV (Control) all over the study period. * Statistical significant difference P < 0.05. ** Highly statistical significant difference P < 0.001.
M.M. El-Tonsy et al. / Acta Tropica 128 (2013) 542–547
carcinogenic effect of DEN since dysplastic changes appeared with a more aggressive nature, and with a smaller dose of DEN in Group II (DEN + Infection) compared to Group I (DEN). Also, serum levels of different biomarkers showed earlier significant statistical difference in Group II (DEN + Infection) than in Group I (DEN) when compared to the Group IV (Control). Meanwhile, S. mansoni infection failed to initiate any dysplastic changes and the serum level of different biomarkers of Group III (Infection) did not show any significant difference compared to the control group throughout the study period. These experimental results agree with Ishii et al. (1994) who found that hepatocellular carcinoma appeared early and in large numbers of mice infected with S. japonicum and given a known carcinogen, compared to mice given the carcinogen only. He concluded that there might be a cause–result relationship between schistosomiasis japonicum and hepatocellular carcinoma. The association of S. mansoni infection and hepatocellular carcinoma was also recorded in a chimpanzee in West Africa. Hepatitis B virus and hepatitis C virus were excluded by serological test in that animal (Abe et al., 1993). From the present study, it is concluded that S. mansoni accelerates hepatic dysplastic changes in the presence of any other risk factor making the cancer appear early and with a more aggressive nature, compared to the same risk in absence of schistosomiasis. Therefore, patients with chronic schistosomal hepatosplenomegaly, especially those who acquired infection in childhood and whose schistosomiasis insult will have the chance to affect their livers for a longer period of time, must be followed up for fear of earlier development of HCC. References Abdel-Rahim, A.Y., 2001. Parasitic infections and hepatic neoplasia. Digestive Diseases 19, 288–291. Abe, K., Kagei, N., Teramura, Y., Ejima, H., 1993. Hepatocellular carcinoma associated with chronic Schistosoma mansoni infection in a chimpanzee. Journal of Medical Primatology 22, 237–239. Anwar, W.A., Khaled, H.M., Amra, H.A., El-Nezami, H., Loffredo, C.A., 2008. Changing pattern of hepatocellular carcinoma (HCC) and its risk factors in Egypt: possibilities for prevention. Mutation Research 659, 176–184. Buchmann, A., Bauer-Hofmann, R., Mahr, J., Norman, R., Schwarz, M., 1991. Mutational activation of c-Ha-ras gene in liver tumors of different rodent strains: correlation with susceptibility to hepatocarcinogenesis. Proceedings of the National Academy of Sciences 88, 911–915. Crawford, J.M., 1990. Pathologic assessment of liver cell dysplasia and benign liver tumors: differentiation from malignant tumors. Seminars in Diagnostic Pathology 7, 115–128. El-Serag, H.B., 2002. Hepatocellular carcinoma: an epidemiologic view. Journal of Clinical Gastroenterology 35, S72–S78. Engall, E., Van Vunakis, H., Langone, J.J., 1980. Methods in Enzymology, 70. Academic Press, New York, pp. 419–492. Gameel, M., El Assaly, N., Medany, H., El-Ashry, N., Mostafa, I., Abd El Raof, E., ElRasky, M., 2009. Evaluation of tumor markers panel in detection of HCC in HCV Egyptian patients. Research Journal of Medicine and Medical Sciences 4, 402–410.
547
Ganne-Carrie, N., Chastang, C., Chapel, E., Munz, C., Pateron, D., Sibony, M., Deny, P., Trinchet, J., Callard, P., Guettier, C., Beaugrand, M., 1996. Predictive score for the development of hepatocellular carcinoma and additional value of liver large cell dysplasia in western patients with cirrhosis. Hepatology 23, 1112–1118. Gomaa, A.I., Khan, S.A., Leen, E.L., Waked, I., Taylor-Robinson, S.D., 2009. Diagnosis of hepatocellular carcinoma. World Journal of Gastroenterology 15, 1301–1314. Habib, S.L., Said, B., Ahmed, T., Awad, M.D., Mostafa, H., Ronald, C., 2006. Novel adenine adducts, N7-guanine-AFB1 adducts, and p53 mutations in patients with schistosomiasis and aflatoxin exposure. Cancer Detection and Prevention 30, 491–498. Harlow, E., Lane, D., 1988. Sampling serum. In: Harlow, E., Lane, D. (Eds.), Antibodies: a Laboratory Manuals, 5, 1st ed. Cold Spring Harbor Laboratory, pp. 116–129. Ishii, A., Matsuoka, H., Aji, T., Ohta, N., Arimoto, S., Wataya, Y., Hayatsu, H., 1994. Parasitic infection and cancer: with special emphasis on Schistosoma japonicum infection (Trematoda). A review. Mutation Research 305, 273–281. Kamal, S., Madwar, M., Bianchi, L., Tawil, A.E., Fawzy, R., Peters, T., Rasen, J.W., 2000. Clinical virological and histopathological features: long-term follow-up in patients with chronic hepatitis C co-infected with S. mansoni. Liver 20, 281–289. Khurana, S., Dubey, M.L., Malla, N., 2005. Association of parasitic infections and cancers. Indian Journal of Medical Microbiology 23, 74–79. Le Bail, B., Bernard, P.H., Carles, J., Balabaud, C., Bioulac-Sage, P., 1997. Prevalence of liver cell dysplasia and association with HCC in a series of 100 cirrhotic liver explants. Journal of Hepatology 21, 835–842. Lee, R.G., Tsamandas, A.C., Demetris, A.J., 1997. Large cell change (liver cell dysplasia) and hepatocellular carcinoma in cirrhosis: matched case–control study, pathological analysis, and pathogenic hypothesis. Hepatology 26, 1415–1422. Liu, H., Wang, Y., Gui, S., 2002. The point mutation of p53 gene in HCC from Anhui Province a non HCC prevalence area in China. World Journal of Gastroenterology 3, 480–482. Nakano, S., Kumada, T., Sugiyama, K., Watahiki, H., Takeda, L., 2008. Clinical significance of serum ferritin determination for hepatocellular carcinoma. American Journal of Gastroenterology 79, 623–627. Ohshima, H., Bartsch, H., 1994. Chronic infections and inflammatory processes as cancer risk factors: possible role of nitric oxide in carcinogenesis. Mutation Research 305, 253–264. Pisani, P., Parkin, D.M., Munoz, N., Ferlay, J., 1997. Cancer and infection: estimates of the attributable fraction in 1990. Cancer Epidemiology, Biomarkers and Prevention 6, 387–400. Pleguezuelo, M., Lopez, L.M., Rodrigues, A., Montero, J.L., Briceno, J., Ciria, R., Muntane, J., de la Mata, M., 2010. Proteomic analysis for developing new biomarkers of hepatocellular carcinoma. World Journal of Hepatology 2, 127–135. Smithers, S.R., Terry, R.J., 1965. The infection of laboratory hosts with cercariae of Schistosoma mansoni and the recovery of adult worms. Parasitology 55, 695–700. Steinmann, P., Keiser, J., Bos, R., Tanner, M., Utzinger, J., 2006. Schistosomiasis and water resources development: systematic review meta-analysis, and estimates of people at risk. Lancet Infectious Diseases 6, 411–425. Strickland, G.T., 2006. Liver disease in Egypt: hepatitis C superseded schistosomiasis as a result of iatrogenic and biological factors. Hepatology 43, 915–922. Thomas, R.M., Berman, J.J., Yetter, R.A., Moore, G.W., Hutchins, G.M., 1992. Liver cell dysplasia: a DNA aneuploid lesion with distinct morphologic features. Human Pathology 23, 496–503. Vennervald, B.J., Polman, K., 2009. Helminths and malignancy. Parasite Immunology 31, 686–696. Watanabe, S., Okita, K., Harada, T., Kodama, T., Numa, Y., Takemoto, T., Takahashi, T., 1983. Morphologic studies of the liver cell dysplasia. Cancer 51, 2197–2205. White, D., Kramer, D., Johnson, G., Dick, F., Hamilton, H., 1986. Enzyme immunoassay (microtiter strips) for the quantitative determination of ferritin in human serum. American Journal of Clinical Pathology 72, 346–349. Yosry, A., 2006. Schistosomiasis and neoplasia. Contributions to Microbiology 13, 81–100. Zhou, L., Liu, J., Luo, F., 2006. Serum tumor markers for detection of hepatocellular carcinoma. World Journal of Gastroenterology 12, 1175–1181.