Functional Activities and Immunohistochemical Cellular Distribution of Glutathione S-Transferases in Normal, Dysplastic, and Squamous Cell Carcinoma Human Oral Tissues

Clinical data show a strong correlation between tobacco and alcohol use and the development of oral squamous cell carcinoma (SCC). While this association implies that the oral mucosa actively metabolizes carcinogens, there is little information which depicts the carcinogen metabolizing enzymes within the oral cavity. Glutathione S-transferases (GSTs) primary function is to detoxify carcinogens by increasing their water solubility, GSTs represent key carcinogen metabolizing enzymes. Notably, individuals with a null phenotype for certain GST isoforms are at an increased risk to develop cancer. This study investigated the function and distribution of GSTs in human oral tissues. Our results from this pilot study showed a trend towards higher GST activities in SCC tissues relative to normal mucosa. Also, relative to normal tissues, the SCC and epithelial dysplasia samples showed a more intense and uniform GST intracellular distribution. GST activities are increased in many high grade cancers. Similarly, our data suggest that GST upregulation occurs in at least a subset of precancerous and malignant oral lesions

Inhibition of Pro-inflammatory and Anti-apoptotic Biomarkers during Experimental Oral Cancer Chemoprevention by Dietary Black Raspberries

Oral cancer continues to be a significant public health problem worldwide. Recently conducted clinical trials demonstrate the ability of black raspberries (BRBs) to modulate biomarkers of molecular efficacy that supports a chemopreventive strategy against oral cancer. However, it is essential that a preclinical animal model of black raspberry (BRB) chemoprevention which recapitulates human oral carcinogenesis be developed, so that we can validate biomarkers and evaluate potential mechanisms of action. We therefore established the ability of BRBs to inhibit oral lesion formation in a carcinogen-induced rat oral cancer model and examined potential mechanisms. F344 rats were administered 4-nitroquinoline 1-oxide (4NQO) (20 µg/ml) in drinking water for 14 weeks followed by regular drinking water for 6 weeks. At week 14, rats were fed a diet containing either 5 or 10% BRB, or 0.4% ellagic acid (EA), a BRB phytochemical. Dietary administration of 5 and 10% BRB reduced oral lesion incidence and multiplicity by 39.3 and 28.6%, respectively. Histopathological analyses demonstrate the ability of BRBs and, to a lesser extent EA, to inhibit the progression of oral cancer. Oral lesion inhibition by BRBs was associated with a reduction in the mRNA expression of pro-inflammatory biomarkers Cxcl1, Mif, and Nfe2l2 as well as the anti-apoptotic and cell cycle associated markers Birc5, Aurka, Ccna1, and Ccna2. Cellular proliferation (Ki-67 staining) in tongue lesions was inhibited by BRBs and EA. Our study demonstrates that, in the rat 4NQO oral cancer model, dietary administration of BRBs inhibits oral carcinogenesis via inhibition of pro-inflammatory and anti-apoptotic pathways

Impact of Cyanotoxin Ingestion on Liver Cancer Development Using an At-Risk Two-Staged Model of Mouse Hepatocarcinogenesis

Exposure to cyanobacterial hepatotoxins has been linked to the promotion and increased incidence of liver cancer in pre-clinical and epidemiologic studies. The family of hepatotoxins, microcystins (MCs), are produced by over 40 cyanobacterial species found in harmful algal blooms (HABs) worldwide, with MC-LR being the most common and potent MC congener. In the current study, we hypothesized that the low-dose chronic ingestion of Microcystis cyanotoxins via drinking water would promote liver carcinogenesis in pre-initiated mice. Four groups of C3H/HeJ mice received one intraperitoneal (i.p.) injection of diethylnitrosamine (DEN) at 4 weeks of age. Three weeks later, the mice were administered ad libitum drinking water containing one of the following: (1) reverse osmosis, deionized water; (2) water containing 500 mg/L phenobarbital (PB500); (3) water with purified MC-LR (10 µg/L) added; or (4) water containing lysed Microcystis aeruginosa (lysate; 10 µg/L total MCs). The exposure concentrations were based on environmentally relevant concentrations and previously established Ohio EPA recreational water MC guidelines. Throughout the 30-week exposure, mouse weights, food consumption, and water consumption were not significantly impacted by toxin ingestion. We found no significant differences in the number of gross and histopathologic liver lesion counts across the treatment groups, but we did note that the PB500 group developed lesion densities too numerous to count. Additionally, the proportion of lesions classified as hepatocellular carcinomas in the MC-LR group (44.5%; p < 0.05) and lysate group (55%; p < 0.01) was significantly higher compared to the control group (14.9%). Over the course of the study, the mice ingesting the lysate also had a significantly lower survival probability (64.4%; p < 0.001) compared to water (96.8%), PB500 (95.0%), and MC-LR (95.7%) exposures. Using cyanotoxin levels at common recreational water concentration levels, we demonstrate the cancer-promoting effects of a single cyanotoxin MC congener (MC-LR). Furthermore, we show enhanced hepatocarcinogenesis and significant mortality associated with combinatorial exposure to the multiple MCs and bioactive compounds present in lysed cyanobacterial cells—a scenario representative of the ingestion exposure route, such as HAB-contaminated water and food

Impact of Microcystin-LR on Liver Function Varies by Dose and Sex in Mice

Microcystin (MC) exposure is an increasing concern because more geographical locations are covered with cyanobacterial blooms as eutrophication and bloom-favoring environmental factors become more prevalent worldwide. Acute MC exposure has been linked to gastrointestinal distress, liver toxicity, and death in extreme circumstances. The goal of this study was to provide an accurate and comprehensive description of MC-LRs impacts on liver pathology, clinical chemistry, and gap junction intercellular communication (GJIC) in CD-1 male and female mice. Mice were exposed to 0, 3000, and 5000/4000 µg/kg/day MC-LR, daily for 7 days, and were necropsied on Day 8. Blood samples for clinical chemistry analysis were processed to serum, while liver sections were fixed for histopathology or evaluated for GJIC using fluorescent cut-load dye. Results show a dose-dependent relationship with MC-LR exposure and hepatocellular hypertrophy, degradation, and necrosis. Clinical chemistry parameters alanine aminotransferase, aspartate aminotransferase, alkaline phosphatase, total bilirubin, and cholesterol increased significantly in MC-LR exposed mice. Clinical chemistry parameter analysis showed significantly increased susceptibility to MC-LR in females compared to males. Changes in GJIC were not noted, but localization of hepatotoxicity near the central veins and midlobular areas was seen. Future toxicity studies involving MCs should consider response differences across sexes, differing MC congeners, and combinatorial exposures involving other cyanotoxins

Cytochromes P450 are Expressed in Proliferating Cells in Barrett's Metaplasia

The expression of cytochromes P450 (CYP) in Barrett's esophagus and esophageal squamous mucosa was investigated. Esophagectomy specimens from 23 patients were examined for CYP expression of CYP1A2, CYP3A4, CYP2C9/10, and CYP2E1 by immunohistochemical analysis, and the expression of CYP1A1, CYP3A4, CYP1B1, CYP2E1, and CYP2C9/10 in these tissues was further confirmed by reverse transcription polymerase chain reaction. Immunohistochemical analysis of esophageal squamous mucosa (n = 12) showed expression of CYP1A2, CYP3A4, CYP2E1, and CYP2C9/10 proteins, but it was noted that cells within the basal proliferative zone did not express CYPs. Immunohistochemical analysis of Barrett's esophagus (n = 13) showed expression of CYP1A2, CYP3A4, CYP2E1, and CYP2C9/10 that was prominent in the basal glandular regions, which are areas containing a high percentage of actively proliferating cells. Immunohistochemical staining for both proliferating cell nuclear antigen and the CYPs further supported the colocalization of CYP expression to areas of active cell proliferation in Barrett's esophagus, whereas in the esophageal squamous epithelium, CYP expression is limited to cells that are not proliferating. RT-PCR with amplification product sequence analysis confirmed CYP1A1, CYP3A4, CYP1B1, CYP2E1, and CYP2C9/10 mRNA expression in Barrett's esophagus. These data suggest that the potential ability of cells in Barrett's esophagus to both activate carcinogens and proliferate may be important risk factors affecting carcinogenesis in this metaplastic tissue