Molecular epidemiology and clinical characteristics of hepatitis D virus infection in Canada.

Background & Aims: HDV affects 4.5-13% of chronic hepatitis B (CHB) patients globally, yet the prevalence of HDV infection in Canada is unknown. To investigate the prevalence, genotype, demographics, and clinical characteristics of HDV in Canada, we conducted a retrospective analysis of (1) HDV antibody and RNA positivity among referred specimens, and (2) a cross-sectional subset study of 135 HDV seropositive +/-RNA (HDV+) patients compared with 5,132 HBV mono-infected patients in the Canadian HBV Network. Methods: Anti-HDV IgG-positive specimens collected between 2012 and 2019 were RNA tested and the genotype determined. Patients enrolled in the Canadian HBV Network were >18 years of age and HBsAg-positive. Clinical data collected included risk factors, demographics, comorbidities, treatment, fibrosis assessment, and hepatic complications. Results: Of the referred patients, 338/7,080 (4.8%, 95% CI 4.3-5.3) were HDV seropositive, with 219/338 RNA-positive (64.8%, 95% CI 59.6-69.7). The HDV+ cohort were more likely to be born in Canada, to be White or Black/African/Caribbean than Asian, and reporting high-risk behaviours, compared with HBV mono-infected patients. Cirrhosis, complications of end-stage liver disease, and liver transplantation were significantly more frequent in the HDV+ cohort. HDV viraemia was significantly associated with elevated liver transaminases and cirrhosis. Five HDV genotypes were observed among referred patients but no association between genotype and clinical outcome was detected within the HDV+ cohort. Conclusions: Nearly 5% of the Canadian HBV referral population is HDV seropositive. HDV infection is highly associated with risk behaviours and both domestic and foreign-born patients with CHB. HDV was significantly associated with progressive liver disease highlighting the need for increased screening and surveillance of HDV in Canada. Lay summary: Evidence of HDV infection was observed in approximately 5% of Canadians who were infected with HBV referred to medical specialists. HDV-positive patients were more likely to be male, born in Canada, or White or Black/African/Caribbean compared to Asian, and to have reported high-risk activities such as injection or intranasal drug use or high-risk sexual contact compared with patients infected with only HBV. Patients infected with HDV were also more likely to suffer severe liver disease, including liver cancer, compared with HBV mono-infected patients.

Comparison of growth features and cancer stem cell prevalence in intrahepatic and extrahepatic cholangiocarcinoma cell lines.

Aim of the study: Intra- and extrahepatic cholangiocarcinoma (I-CCA and E-CCA respectively) exhibit different growth features that contribute to different clinical outcomes. Cancer stem cells (CSCs) influence tumor growth and thereby may be responsible for these differences. The aim of this study was to document and compare the growth features of human I-CCA and E-CCA cell lines and determine whether any differences observed could be explained by differences in the prevalence and/or stem cell surface marker (SCSM) expression profiles of CSCs within the tumor cell lines. Material and methods: Six CCA cells lines, three I-CCA and three E-CCA, were studied. Tumor cell growth features including cell proliferation, colony/spheroid formation, migration and invasion were documented. CSC prevalence and SCSM expression profiles were examined by flow cytometry. Results: I-CCA cells had significantly increased proliferative activity, shorter doubling times and were more invasive than E-CCA cells, while colony/spheroid formation and migration were similar in the two cell populations. There were no significant differences in CSC prevalence rates or SCSM expression profiles. Conclusions: These findings suggest that I-CCA cells proliferate at a more rapid rate and are more invasive than E-CCA cells but the differences cannot be explained by differences in the prevalence or SCSM expression profiles of CSCs within the tumor cell population.

Fibroblast Growth Factor 19 Induced Changes in Non-malignant Cholangiocytes.

BACKGROUND AND AIMS: Fibroblast growth factor (FGF)19 has been implicated in the pathogenesis of murine hepatocellular carcinoma. Whether it plays a role in the development or course of human cholangiocarcinoma remains to be determined. The aim of this study was to determine whether prolonged exposure to FGF19 results in the transformation of non-malignant human cholangiocytes into cells with malignant features. METHODS: Human SV-40 transfected non-malignant H69 cholangiocytes were cultured with FGF19 (0-50 ng/mL) for 6 weeks, followed by 6 weeks with medium alone. Cell proliferation, invasion, stem cell surface markers, oncofetoprotein expression, state of differentiation, epithelial-mesenchymal transition (EMT) and interleukin (IL)-6 expression were documented at various time intervals throughout the 12-week period. RESULTS: FGF19 exposure was associated with significant increases in cell proliferation, de-differentiation, EMT and IL-6 expression. However, each of these effects returned to baseline or control values during the 6-week FGF19 free follow-up period. The remaining cell properties remained unaltered. CONCLUSIONS: Six weeks of FGF19 exposure did not result in the acquisition of permanent malignant features in non-malignant, human cholangiocytes.

Hepatic adenomatosis in a young woman with non-familial maturity-onset diabetes of the young type 3.

Hepatic adenomatosis (HA) is a rare condition in which multiple adenomas exist in an otherwise healthy liver. The most common subtype (H-HA) is associated with bi-allelic, somatic hepatic nuclear factor 1-alpha (HNF1A) mutations. Maturity-onset diabetes of the young type 3 (MODY3) is most often seen in young individuals with heterozygous, germline mutations in HNF1A. In this report, we describe a 17-year-old woman with H-HA and non-familial MODY3.

Alterations in chemokine receptor CCR5 activity influence tumor cell biology in human cholangiocarcinoma cell lines.

INTRODUCTION AND OBJECTIVES: Intrahepatic (I-CCA) and extrahepatic (E-CCA) cholangiocarcinoma (CCA) have different growth patterns and risks for tumor metastasis. Inhibition and/or activation of the chemokine receptor CCR subclasses have been reported to alter tumor cell biology in non-CCA cancers. In this study we documented CCR expression profiles in representative human I-CCA and E-CCA cell lines and the in vitro effects of CCR antagonists and agonists on tumor cell biology. MATERIALS AND METHODS: CCR expression profiles were documented by real-time reverse transcription polymerase chain reaction; cell proliferation by WST-1; spheroid formation by sphere dimensions in anchorage-free medium; cell migration by wound healing and invasion by Transwell invasion chambers. RESULTS: All 10 CCR motifs (CCR1-10) were expressed in the I-CCA, HuCCT1 cell line and six (CCR4, 5, 6, 8, 9 and 10) in the E-CCA, KMBC cell line. In HuCCT1 cells, CCR5 expression was most abundant whereas in KMBC cells, CCR6 followed by CCR5 were most abundant. The CCR5 antagonist Maraviroc significantly inhibited cell proliferation, migration and invasion in HuCCT1 cells, and spheroid formation and invasion in KMBC cells. The CCR5 agonist RANTES had no effect on HuCCT1 cells but increased cell proliferation, migration and invasion of KMBC cells. CONCLUSION: These results suggest that CCR expression profiles differ in I-CCA and E-CCA. They also indicate that CCR5 antagonists and agonists have cell-specific effects but in general, CCR5 inactivation inhibits CCA tumor cell aggressiveness. Additional research is required to determine whether CCR5 inactivation is of value in the treatment of CCA in humans.

Enhanced gemcitabine cytotoxicity with knockdown of multidrug resistance protein genes in human cholangiocarcinoma cell lines.

BACKGROUND AND AIM: Cholangiocarcinoma (CCA) is an often fatal primary cancer of the liver that tends to be resistant to chemotherapy. Multidrug resistance proteins (MRPs) contribute to the chemoresistance of these tumors. The objectives of the study were to document MRP expression profiles in two representative human intrahepatic and extrahepatic CCA cells lines (HuCCT1 and KMBC, respectively) and gemcitabine-induced cytotoxicity prior to and following MRP knockdown. METHODS: Multidrug resistance protein mRNA and protein expression were documented by real-time reverse transcription-polymerase chain reaction and western blots, respectively. MRP knockdown was achieved with lentivirus small hairpin RNA constructs. RESULTS: Prior to gemcitabine exposure, MRP1, MRP2, MRP4, MRP5, and MRP6 mRNA were expressed in HuCCT1 cells and MRP1, MRP3, MRP4, and MRP5 in KMBC cells. Following gemcitabine exposure, MRP5 and MRP6 expressions were significantly upregulated in HuCCT1 cells and MRP5 in KMBC cells. In HuCCT1 cells, although MRP5 knockdown had no effect, MRP6 knockdown significantly increased gemcitabine-induced cytotoxicity. In KMBC cells, MRP5 knockdown significantly increased gemcitabine cytotoxicity. CONCLUSIONS: Inhibition of MRP6 expression in intra-hepatic and MRP5 in extra-hepatic should be explored as potential treatments for CCA in humans.

A lack of association between increases in gastrointestinal, liver, and urologic cancers and cyanobacterial contamination of fresh water lakes in Manitoba.

Cyanobacteria or blue-green algae are becoming increasingly abundant in North American fresh water lakes. Toxins produced by cyanobacteria have been associated with gastrointestinal injury, liver failure, and nephrotoxicity. They have also been implicated in the pathogenesis of gastrointestinal and liver cancers. The purpose of the present study was to determine whether the incidence rates of gastrointestinal, liver, and urologic cancers are increasing in the province of Manitoba and, if so, whether these increases spatially and/or temporally correlate with areas where cyanobacterial contamination of fresh water lakes have been identified. Cancer incidence data were obtained from the Manitoba Cancer Registry. Cyanobacterial contamination data, as reflected by microcystin toxin concentrations, were available from the Manitoba Water Stewardship. ArcGIS mapping was employed to document spatial and temporal relationships between cancer incidence and cyanobacterial data. The results revealed that although the incidence rates for all three cancers have increased over the past 20-25 years, these increases were not disproportionally higher in zones with high microcystin toxin determinations. The results of this study argue against increased exposure to cyanotoxins as an explanation for the increase in gastrointestinal, liver and urologic cancers in Manitoba.

The effects of hyperthermia on human hepatocellular carcinoma stem and mature cancer cells.

INTRODUCTION AND OBJECTIVES: Hepatocellular carcinoma (HCC) can recur following radiofrequency ablation and other hyperthermic treatment modalities. Cancer stem cells (CSCs) are a subpopulation of HCC cells that are difficult to eradicate and largely responsible for tumor recurrences. Thus, the principal objective of this study was to determine whether human HCC CSCs are relatively thermal-resistant compared to non-stem or mature cancer cells (MCCs). MATERIALS AND METHODS: Epithelial cell adhesion molecule (EpCAM) positive enriched CSCs and EpCAM- MCCs were derived from a human HCC cell line using fluorescence activated cell sorting. Each cell population was exposed to 65°C heat for 0-16min and survival documented at various time points. RESULTS: Cell survival curves were similar in CSC and MCCs throughout the 16min heat exposure period. Maximum killing was obtained after 12-14min of heat exposure. Cytoprotective, heat shock proteins-70 (HSP70) and -90 (HSP90) mRNA expression were not disproportionately increased in CSCs. CONCLUSIONS: These results suggest that human HCC CSCs are not more thermal resistant than MCCs and therefore, do not support the hypothesis that HCC recurrences following hyperthermic treatment reflect CSC thermal-resistance.

Hepatitis B Virus Infection of Normal Hepatic Stem/Progenitor Cells.

BACKGROUND/AIMS: Whether the hepatitis B virus (HBV) infects normal hepatic stem/progenitor cells (NSCs) and if so, whether such infections play a role in the pathogenesis of HBV-induced chronic liver disease (CLD) and/or hepatocellular carcinoma (HCC) remains to be determined. The objectives of this study were to determine whether HBV infects NSCs and whether such infections alter NSC activity in a manner likely to contribute to the development of CLD and/or HCC. METHODS: Liver biopsies from five hepatitis B surface antigen (HBsAg) positive patients were co-stained for HBcAg and HBx and the stem cell markers EpCAM, Oct-4 and Nanog. In addition, primary NSCs derived from healthy human livers were exposed to HBV contaminated serum in vitro. Supernatant and/or cellular HBsAg, HBcAg and HBV-DNA expression were documented over the subsequent 30 days of culture. Pro- and anti-inflammatory cytokine expression, membrane potential differences (PDs), proliferative and telomerase activities of HBV-infected NSCs were also documented. RESULTS: Markers of HBV infection were present within the NSC population of all five biopsy specimens. In vitro, HBV markers appeared within three days of exposure, peaked in expression after 10-15 days and remained positive thereafter for the duration of cell viability. There were no consistent changes in HBV-infected NSC pro- or anti-inflammatory cytokine expression, membrane PDs, proliferative or telomerase activities. CONCLUSIONS: Although the results of this study need to be confirmed, they suggest that HBV infects human NSCs but in the short term, do not alter those NSC features or activities associated with CLD and/or HCC.

The effects of Ciji-Hua'ai-Baosheng on immune function of mice with H22 hepatocellular carcinoma receiving chemotherapy.

BACKGROUND: Ciji-Hua'ai-Baosheng Decoction (CHBD) is a traditional Chinese formula that may attenuate the toxicity and side-effects of chemotherapy. The formula may also prolong the life of cancer patients. Whether CHBD should be employed as adjunctive therapy for cancer patients receiving chemotherapy has yet to be determined as does the mechanism whereby CHBD exerts its beneficial effects. AIM OF THE STUDY: To document the potential effects of CHBD on tumor growth and immune function in a murine model of hepatocellular carcinoma (HCC) receiving chemotherapy. MATERIALS AND METHODS: Sixty Kunming mice were injected subcutaneously with H22 hepatoma cells in the right anterior armpit. After seven days, the mice with formed tumors were injected with Cytoxan (CTX) (200 mg/kg) to establish the chemotherapy model. These mice were randomly divided into 5 groups: model (untreated controls), control (CTX,33.33 mg/kg), and high CHBD (H) (117 g/kg), moderate CHBD (M) (58.5 g/kg) and low CHBD (L) (29.25 g/kg) treated groups. Tumor weights and inhibitory ratio (decrease in tumor dimensions), histology of tumor, colon, spleen and liver, and biochemical tests of liver and kidney function were documented after 10 days. Serum and tumor IL-2, IFN-γ, IL-6, and TNF-α levels were determined by enzyme-linked immunosorbent assay (ELISA) and Western blot respectively. The potential bioactive compounds in CHBD were characterized by UHPLC-MS. RESULTS: Although tumor weights were decreased in CTX alone and CHBD (H) and CHBD (M) groups (-66%, -41% and -25% respectively), tumor cell density was reduced to the greatest extent in the CHBD (H) group. CHBD had no evident effects on liver and kidney function. CTX-induced colon inflammation and decrease in spleen lymphocytes were attenuated with CHBD treatment. CHBD increased serum IL-2, IFN-γ and TNF-α, but decreased IL-6 levels in serum and tumor tissue. UHPLC-MS analysis of CHBD revealed the presence of 11 bioactive compounds. CONCLUSIONS: In this murine model of HCC receiving chemotherapy, CHBD inhibited tumor growth, improved immune function and pro-inflammatory cytokine responses while attenuating CTX-associated side effects.

Cancer Stem Cells are Depolarized Relative to Normal Stem Cells Derived from Human Livers

ABSTRACT Introduction and aim. The inability to distinguish cancer (CSCs) from normal stem cells (NSCs) has hindered attempts to identify safer, more effective therapies for hepatocellular carcinoma (HCC). The aim of this study was to document and compare cell membrane potential differences (PDs) of CSCs and NSCs derived from human HCC and healthy livers respectively and determine whether altered GABAergic innervation could explain the differences. Material and methods. Epithelial cell adhesion molecule (EpCAM) positive stem cells were isolated from human liver tissues by magnetic bead separations. Cellular PDs were recorded by microelectrode impalement of freshly isolated cells. GABAA receptor subunit expression was documented by reverse transcriptase polymerase chain reaction (RT-PCR) and immunofluorescence. Results. CSCs were significantly depolarized (-7.0 ± 1.3 mV) relative to NSCs (-23.0 ± 1.4 mV, p < 0.01). The depolarized state was associated with different GABAA receptor subunit expression profiles wherein phasic transmission, represented by GAGAA α3 subunit expression, was prevalent in CSCs while tonic transmission, represented by GABAA α6 subunit expression, prevailed in NSCs. In addition, GABAA subunits α3, β3, γ3 and δ were strongly expressed in CSCs while GABAA π expression was dominant in NSCs. CSCs and NSCs responded similarly to GABAA receptor agonists (ΔPD: 12.5 ± 1.2 mV and 11.0 ± 3.5 mV respectively). Conclusion. The results of this study indicate that CSCs are significantly depolarized relative to NSCs and these differences are associated with differences in GABAA receptor subunit expression. Together they provide new insights into the pathogenesis and possible treatment of human HCC.

Cancer Stem Cells are Depolarized Relative to Normal Stem Cells Derived from Human Livers.

INTRODUCTION AND AIM: The inability to distinguish cancer (CSCs) from normal stem cells (NSCs) has hindered attempts to identify safer, more effective therapies for hepatocellular carcinoma (HCC). The aim of this study was to document and compare cell membrane potential differences (PDs) of CSCs and NSCs derived from human HCC and healthy livers respectively and determine whether altered GABAergic innervation could explain the differences. MATERIAL AND METHODS: Epithelial cell adhesion molecule (EpCAM) positive stem cells were isolated from human liver tissues by magnetic bead separations. Cellular PDs were recorded by microelectrode impalement of freshly isolated cells. GABAA receptor subunit expression was documented by reverse transcriptase polymerase chain reaction (RT-PCR) and immunofluorescence. RESULTS: CSCs were significantly depolarized (-7.0 ± 1.3 mV) relative to NSCs (-23.0 ± 1.4 mV, p &lt; 0.01). The depolarized state was associated with different GABAA receptor subunit expression profiles wherein phasic transmission, represented by GAGAA α3 subunit expression, was prevalent in CSCs while tonic transmission, represented by GABAA α6 subunit expression, prevailed in NSCs. In addition, GABAA subunits α3, ß3, ϒ3 and δ were strongly expressed in CSCs while GABAA π expression was dominant in NSCs. CSCs and NSCs responded similarly to GABAA receptor agonists (ΔPD: 12.5 ± 1.2 mV and 11.0 ± 3.5 mV respectively). CONCLUSION: The results of this study indicate that CSCs are significantly depolarized relative to NSCs and these differences are associated with differences in GABAA receptor subunit expression. Together they provide new insights into the pathogenesis and possible treatment of human HCC.

Diagnostic considerations for cholestatic liver disease.

Cholestatic liver disease results from insufficient bile synthesis, secretion and/or flow through the biliary tract. Common presenting features include fatigue, pruritus, and cholestatic liver enzyme abnormalities wherein elevations of serum alkaline phosphatase and gamma-glutamyltransferases levels exceed those of alanine and aspartate aminotransferases. With prolonged cholestasis, fat soluble vitamin deficiencies, fibrosis, cirrhosis, and, on occasion, carcinoma of the biliary tract or liver can occur. Once mechanical obstruction to bile flow has been ruled out, the majority of causes can be classified as immune-mediated, infectious, or miscellaneous. Because specific therapeutic options are increasing for many causes of cholestasis, an accurate diagnosis is an important first step towards treatment. Thus, this review focuses on the diagnostic features of non-mechanical causes of cholestasis.

Evidence of Hepatitis B Virus Infection in Cancer and Noncancer Stem Cells Associated with Human Hepatocellular Carcinoma.

Both the hepatitis B virus (HBV) and cancer stem cells (CSCs) have been independently implicated in the pathogenesis of hepatocellular carcinoma (HCC). To date, there have been no reports describing HBV infection within CSCs. In this report we describe HBV core (HBcAg) and HBx protein expression within CSCs associated with human HCC. HBV markers were also identified in nonmalignant stem cells present in adjacent nontumor tissue. These findings provide new insights into the pathogenesis of HBV-induced HCC and are potentially relevant to the treatment of both HCC and chronic HBV.

The combination effects of Shen-Ling-Bai-Zhu on promoting apoptosis of transplanted H22 hepatocellular carcinoma in mice receiving chemotherapy.

ETHNOPHARMACOLOGICAL RELEVANCE: Shen-Ling-Bai-Zhu Powder (SLBZP) is a classic traditional Chinese medical formula that has been used for several decades in the treatment of patients with gastrointestinal malignancies. Whether SLBZP is best employed as single agent or adjunctive therapy has yet to be determined as does the mechanism whereby SLBZP exerts its anti-tumor effects. AIM OF THE STUDY: To investigate the effects of SLBZP alone and in combination with Cytoxan (CTX) on tumor growth, malignant cell apoptosis and Akt/Nuclear Factor kappa B (NF-КB) signaling in a murine model of hepatocellular carcinoma (HCC) receiving chemotherapy. MATERIALS AND METHODS: Sixty-four adult mice developed HCC following subcutaneous inoculation with H22 hepatocellular carcinoma cells. Seven days later, all received chemotherapy with CTX (200mg/kg) once. Mice were then randomized into eight study groups (N=8/group). Three groups were treated with different concentrations of SLBZP alone (6.00, 3.00, 1.5g/kg), three with SLBZP (6.00, 3.00, 1.5g/kg) plus CTX (20mg/kg), one with CTX (20mg/kg) alone (positive control), and one with physiologic saline (untreated, negative control). All groups were treated for 14 days. Tumor size, histology and serum or tissue levels and/or mRNA expression of PDGF-BB, VEGF, Ang-1, Ang-2, NF-КB, B-cell lymphoma-2 (Bcl-2); B-cell lymphoma-extra large (Bcl-xL); X-linked inhibitor of apoptosis (XIAP), Survivin, Caspase-3, Caspase-9, Caspase-7, Akt and phosphorylated Akt expression were documented at the end of treatment. RESULTS: Compared to untreated negative controls, tumor sizes were decreased in the CTX alone, SLBZP (M)+CTX and SLBZP (H)+CTX groups (-52%,-53% and -58% respectively). Tumor cell density was decreased in all treated groups but most apparent in the SLBZP (H)+CTX group. Electron microscopic evidence of apoptosis was also most apparent in this group. Serum and/or tissue levels and expression of PDGF-BB, VEGF, Ang-1, Ang-2, their downstream signaling proteins and anti-apoptotic markers were lowest and pro-apoptotic markers highest in SLBZP (H)+CTX treated mice. CONCLUSIONS: In this chemotherapy-induced animal model of HCC, SLBZP was most efficacious as adjunctive therapy and appears to act by inhibiting tumor growth promoters and anti-apoptotic proteins while enhancing pro-apoptotic proteins.

The effects of Tao-Hong-Si-Wu on hepatic necroinflammatory activity and fibrosis in a murine model of chronic liver disease.

BACKGROUND: Tao-Hong-Si-Wu decoction (THSWD) is a traditional Chinese herbal medicine that has been used for centuries in the treatment of Chinese patients with chronic liver disease. Recently, THSWD has been reported to alter vascular endothelial growth factor (VEGF) induced angiogenesis, raising the possibility that in addition to its anti-inflammatory properties; THSWD might also inhibit hepatic blood flow associated fibrosis. AIM: To document the effects of THSWD on hepatic necroinflammatory disease activity, fibrosis and VEGF signaling in a murine model of chronic liver disease. METHODS: Sixty adult mice were equally divided into six study groups. Five groups were exposed to subcutaneous carbon tetrachloride (0.1 ml/10 g BW) for six weeks. Three of the five groups were treated with different concentrations of THSWD (4.25, 8.50, 17.00 g/kg), one with 0.1mg/kg of Colchicine (positive control), and one with physiologic saline (negative control). Mice in the sixth group were not exposed to CCl4 and remained untreated (healthy controls). Liver enzymes/function tests, hyaluronic acid and laminin levels were measured in serum, and hepatic histology, VEGF, Flt-1 and kinase insert domain-containing receptor (KDR), Akt and phosphorylated Akt (pAkt) expression were documented in liver tissue at the end of treatment. RESULTS: Hepatic necroinflammatory disease activity and fibrosis were significantly attenuated in THSWD treated mice in a dose dependent manner. These beneficial results were similar and often exceeded those achieved with Colchicine. In addition, VEGF, Flt-1, KDR, Akt and pAkt mRNA and protein expression were reduced in TSHWD treated mice. CONCLUSIONS: In this animal model of chronic liver disease, THSWD decreased hepatic necroinflammatory disease and fibrosis. Inhibition of VEGF expression and downstream signaling were associated with these findings. Further studies with this and other TCMs as treatment for chronic liver disease are warranted.

The Geographic Distribution of Liver Cancer in Canada Does Not Associate with Cyanobacterial Toxin Exposure.

BACKGROUND: The incidence of liver cancer has been increasing in Canada over the past decade, as has cyanobacterial contamination of Canadian freshwater lakes and drinking water sources. Cyanotoxins released by cyanobacteria have been implicated in the pathogenesis of liver cancer. OBJECTIVE: To determine whether a geographic association exists between liver cancer and surrogate markers of cyanobacterial contamination of freshwater lakes in Canada. METHODS: A negative binomial regression model was employed based on previously identified risk factors for liver cancer. RESULTS: No association existed between the geographic distribution of liver cancer and surrogate markers of cyanobacterial contamination. As predicted, significant associations existed in areas with a high prevalence of hepatitis B virus infection, large immigrant populations and urban residences. DISCUSSION AND CONCLUSIONS: The results of this study suggest that cyanobacterial contamination of freshwater lakes does not play an important role in the increasing incidence of liver cancer in Canada.

Long-term, low-dose exposure to microcystin toxin does not increase the risk of liver tumor development or growth in mice.

AIM: Cyanotoxins are biological toxins produced by cyanobacteria (blue green algae) that have been implicated in the pathogenesis of liver tumors. Based on acute toxicity studies, the World Health Organization has designated 1.0 µg/L of cyanotoxin-contaminated drinking water as the safe allowable limit for daily oral consumption. The aim of this study was to determine whether long-term exposure to this concentration of cyanotoxins is capable of initiating or promoting the growth of liver tumors. METHODS: In the present study, four groups of adult, male CD-1 mice (n = 20/group) were exposed to either drinking water alone (water group), drinking water containing 1.0 µg/L of microcystin-LR (MC-LR group), MC-LR plus the tumor promoter thioacetamide (MC-LR/TAA group) or thioacetamide alone (TAA group). Following 28 weeks of exposure, mice were killed and the livers examined for tumor number and size. RESULTS: No tumors were present in the water or MC-LR alone groups while five mice in the MC-LR/TAA group and four in the TAA alone group developed liver tumors. The mean size of the tumors in the MC-LR/TAA and TAA alone groups were similar as were the results of Ki-67 staining, number of atypical mitoses and liver cancer gene expression profiles. In vitro MC-LR (0.1-1000 µg/L) exposure did not induce malignant transformation of WB-F344 hepatic stem cells or increase the proliferative activity or invasiveness of PLC/PRF/5 malignant hepatocytes. CONCLUSION: The results of this study suggest that long-term, low dose cyanotoxin exposure is unlikely to result in liver tumor development or enhance existing tumor growth.

Hepatitis B and hepatitis C viral infections in patients with chronic lymphocytic leukemia.

BACKGROUND: Whether chronic hepatitis B virus (HBV) or hepatitis C virus (HCV) infections contribute to the pathogenesis and/or course of chronic lymphocytic leukemia is unclear. OBJECTIVE: To document the prevalences of HBV and HCV infections in chronic lymphocytic leukemia patients, and to determine whether infected patients experience more aggressive disease than those without infection. METHODS: Patient sera were screened for antibodies to HBV core antigen and HCV (anti-HCV) using ELISA; both sera and peripheral blood lymphocytes were further tested (regardless of antibody results) for HBV-DNA and HCV-RNA using real-time polymerase chain reaction. Prognostic markers for chronic lymphocytic leukemia included Rai stage, IgVH mutational status, ß2-microglobulin levels, Zap-70 and CD38 status. RESULTS: Fourteen of 222 (6.3%) chronic lymphocytic leukemia patients and two of 72 (2.8%) healthy controls tested positive for previous or ongoing HBV infection (OR 2.4 [95% CI 0.5 to 7.7]; P=0.25) while four of 222 (1.8%) chronic lymphocytic leukemia patients and one of 72 (1.4%) controls tested positive for HCV markers (OR 1.3 [95% CI 0.2 to 6.4]; P=0.81). The levels and distribution of the various indicators of aggressive chronic lymphocytic leukemia disease were similar among HBV- and HCV-infected and uninfected patients. Survival times were also similar. Occult HBV and HCV infection (HBV-DNA or HCV-RNA positive in the absence of diagnostic serological markers) were uncommon in chronic lymphocytic leukemia patients (0.5% and 1.8%, respectively). CONCLUSIONS: The results of the present study do not support the hypothesis that HBV or HCV infections play an important role in the pathogenesis or course of chronic lymphocytic leukemia.