Proposal to Eliminate Urethane-Treated Positive Control Dose Groups in 26-Week Tg.rasH2 Carcinogenicity Studies.

Short-term (26 weeks) Tg.rasH2 mouse carcinogenicity studies have been conducted as an alternative model to the conventional 2-year mouse carcinogenicity studies, using urethane as a positive control material. In these studies, urethane was used at a dose of 1,000 mg/kg/dose, administered intraperitoneally on days 1, 3, and 5. Urethane consistently produces lung adenomas and carcinomas and hemangiosarcomas of the spleen, proving validity of the assay. We conducted 3 pilot studies at 3 different sites of Charles River Laboratories using a lower dose of urethane (500 mg/kg/dose), administered on days 1, 3, and 5, followed by a 12-week observation period. Our results demonstrate that a lower dose can be used successfully with fewer number of animals per sex to prove the validity of the assay. However, based on our cumulative experience with this model, we propose to eliminate positive control dose groups in future Tg.rasH2 carcinogenicity studies.

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.

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.

Comparison of phenethyl and 6-phenylhexyl isothiocyanate-induced toxicity in rat esophageal cell lines with and without glutathione depletion.

Phenethyl isothiocyanate (PEITC) and its synthetic homolog, 6-phenylhexyl isothiocyanate (PHITC), are both potent inhibitors of 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK)-induced lung mice tumorigenesis. However, unlike PEITC, PHITC enhanced N-nitrosomethylbenzylamine (NMBA)-induced rat esophageal tumorigenesis. These findings imply that due to its unique chemical properties, PHITC's effects on esophageal cells are procarcinogenic rather than chemopreventive. Relative to PEITC, PHITC is more lipophilic and less reactive, which could result in higher PHITC intracellular levels. Due to ITCs' inherently high level of thiol reactivity, increased intracellular levels of PHITC have the potential to deplete intracellular glutathione (GSH) reserves. Since GSH is a primary intracellular antioxidant and cytoprotective enzyme cofactor, preservation of intracellular GSH status is crucial for cytoprotection. Despite the recognized importance of isothiocyanate structure with the potential for toxicity, no studies have yet investigated the association between the primary intracellular free thiol, GSH, and isothiocyanate-induced toxicity in this target cell population. The present study investigated whether PEITC and PHITC display unique cytotoxic profiles in cultured rat esophageal cells, and also monitored the effects of ITC challenge on cellular GSH status. A final series of experiments investigated the converse i.e., affects of modulation of intracellular GSH status on ITC-mediated toxicity. Dose-response curves revealed that PEITC was significantly more toxic in tumorigenic and non-tumorigenic cells relative to PHITC. The ITC-GSH interaction studies demonstrated comparable GSH levels following either PEITC or PHITC challenge, and also showed that GSH depletion did not augment ITC-mediated cellular toxicity. While our data demonstrate structure related differences in ITC-mediated cytotoxicities, these differences do not appear to be directly attributable to cellular GSH pools.

Endostatin inhibits migration and invasion of head and neck squamous cell carcinoma cells.

Head and neck cancers are significant due to their high morbidity and associated complications. We report, for the first time, that endostatin directly affects epithelial lineage human cells derived from tumors of patients with head and neck squamous cell carcinoma (HNSCC). This study investigated endostatin's effects on several HNSCC cellular functions that are essential for tumor progression. We determined that exposure of HNSCC cells to endostatin activated the transcription-activating factors, NF-xB and AP-1 in a cell-line-dependent fashion. Endostatin also down-regulated the gene expression of several pro-migratory molecules. Migration and invasion assays showed that endostatin significantly inhibited these functions that are essential for tumor progression. Fluorescent labeling studies showed endostatin co-localized to tropomysin-binding HNSCC the microfilaments, suggesting endostatin's suppression of HNSCC cell migration and invasion may reflect perturbation of the microfilament function. Our data imply that endostatin's clinical efficacy extends beyond angiostatic properties to encompass a direct anti-tumorigenic effect against HNSCC cells.

AIDS-related Kaposi's sarcoma cells rapidly internalize endostatin, which co-localizes to tropomysin microfilaments and inhibits cytokine-mediated migration and invasion.

AIDS-related Kaposi's sarcoma (KS) is the most common HIV-related malignancy. In some respects, KS is analogous to other angioproliferative diseases, in that KS lesions are highly vascularized and promoted by inflammatory cytokines. However, unlike other cancers or inflammatory mediated vascular diseases, KS is unique in that the KS lesional cells both express and respond to the complete angiogenic cytokines vascular endothelial growth factor (VEGF) and basic fibroblast growth factor (bFGF). Therefore, the angiogenic phenotype, which is crucial for cancer progression, is inherent to KS tumor cells. Due to the recognized importance of angiogenesis in cancer progression, numerous angiostatic agents are being investigated as potential therapeutic agents. One such agent is endostatin, which is a 20-kDa carboxyl-terminal fragment of collagen XVIII that has demonstrated potent angiostatic activities at both the in vivo and in vitro levels. Since endostatin is recognized as a potent angiostatic agent, the majority of in vitro endostatin studies have evaluated its effects on endothelial cells. Although KS cells are speculated to arise from endothelial cell precursors and KS lesions are highly vascularized, no previous studies have investigated endostatin-KS cell interactions. This present study evaluated endostatin's effects on KS tumor cell: (i) signal transduction (endostatin internalization and transcription factor activation), and (ii) migration and invasion (functional activity assays and tropomysin co-localization). Our results show that KS cells rapidly internalize endostatin and that endostatin initiates activation of the transcription activating factors nuclear factor-kappaB (NF-kappaB) and activating protein 1 (AP-1). Our data also show that internalized endostatin co-localizes to tropomysin microfilaments and acts to inhibit KS cell migration and invasion in response to the clinically relevant angiogenic cytokines VEGF and bFGF. As a consequence of its combined angiostatic and antitumorigenic activities, endostatin could provide dual therapeutic benefits for patients with mucocutaneous KS.

Inhibitory effects of 2-mercaptoethane sulfonate and 6-phenylhexyl isothiocyanate on urinary bladder tumorigenesis in rats induced by N-butyl-N-(4-hydroxybutyl)nitrosamine.

The effects of 2-mercaptoethane sulfonate (MESNA) and phenethyl and 6-phenylhexyl isothiocyanates (PEITC and PHITC) on urinary bladder tumorigenesis were investigated in Wistar rats treated with N-butyl-N-(4-hydroxybutyl)nitrosamine (BHBN). A total of 190 male rats were divided into eight groups. Animals in groups 1, 3 and 4 were administered BHBN in drinking water (0.025%) and group 2 rats were given 0.025% BHBN plus 0.05% MESNA solution for 15 weeks. Rats in groups 3 and 4 were fed a diet supplemented with PEITC or PHITC at a dose level of 0.5 micromol/g for 16 weeks starting 1 week prior to the BHBN treatment. Rats in groups 5-7 served as MESNA, PEITC or PHITC alone controls. Group 8 served as a non-treatment control. The chromatographic profile of drinking water for group 2 animals indicated no chemical interaction between BHBN and MESNA. The incidences of papillomas in the bladder and total bladder tumors including transitional cell carcinomas were significantly (P<0.01) reduced in group 2 as compared to that in group 1. The multiplicities of papillomas or total bladder tumors were significantly (P<0.01) lower in group 2 and 4 rats treated with MESNA and PHITC, respectively, than in group 1 rats. The PEITC treatment, however, did not affect the BHBN-induced bladder tumorigenesis. No bladder tumors were found in animals of groups 5-8. The results in the present study clearly indicate that both MESNA and PHITC are potent chemopreventive agents against bladder tumor development in rats induced by BHBN.

Curcumin activates the aryl hydrocarbon receptor yet significantly inhibits (-)-benzo(a)pyrene-7R-trans-7,8-dihydrodiol bioactivation in oral squamous cell carcinoma cells and oral mucosa.

The development of oral squamous cell carcinoma (SCC) shows a positive correlation with the carcinogen exposure that occurs during tobacco and alcohol use. The purpose of this study was to investigate whether the naturally occurring chemopreventive agent, curcumin, modulates expression and function of carcinogen- metabolizing enzymes in human keratinocytes isolated from oral SCC tumors. Dose-response studies demonstrated that curcumin concentrations of >or=25 micro M were cytotoxic for oral SCC cells. Curcumin increased both expression (reverse transcription-PCR analyses) and function (high-performance liquid chromatography determination of ethoxyresorufin metabolism) of cytochrome P-450 (CYP) 1A1 and/or CYP1B1. The aryl hydrocarbon receptor (AhR), which up-regulates a battery of genes associated with carcinogen metabolism, is activated by polycyclic aromatic hydrocarbons such as the tobacco-associated carcinogen benzo(a)pyrene. Electromobility shift assays demonstrated that similar to the established AhR ligand 2,3,7,8,-tetrachlorodibenzo-p-dioxin, curcumin inclusion resulted in AhR nuclear translocation and formation of the transcriptionally active AhR-aryl hydrocarbon receptor nuclear translocator complex. Cellular capacity to bioactivate the tobacco-associated carcinogen (-)-benzo(a)pyrene-7R-trans-7,8-dihydrodiodiol was determined by evaluating conversion of the carcinogenic metabolite diol epoxide to stable tetrols via high-performance liquid chromatography. Results of our metabolism studies showed that curcumin significantly inhibited CYP1A1-mediated benzo(a)pyrene diol bioactivation in both oral SCC cells and intact oral mucosa. Because CYP1A1 is one of the primary carcinogen-activating enzymes in oral mucosa, the use of curcumin as an oral cavity chemopreventive agent could have significant clinical impact via its ability to inhibit carcinogen bioactivation.

Functional role of cytochrome p-450 2a3 in N-nitrosomethylbenzylamine metabolism in rat esophagus.

Previous in vitro studies demonstrated that the rat esophageal carcinogen N-nitrosomethylbenzylamine (NMBA) is metabolically activated by cytochrome P-450s (CYP) 2A3 and 2E1. However, the in vivo role of these P-450s in the metabolism of NMBA has not been fully evaluated. In this study, the effects of single and multiple doses of NMBA were investigated on CYP2A3 and CYP2E1 mRNA expression in the rat esophagus and lung. Seven- to 8-wk old male Fischer 344 rats were administered a single subcutaneous dose of NMBA at either 0.5 mg/kg or 2 mg/kg body weight, after which the rats were sacrificed at 1, 3, 6, 12, 24, 48, and 72 h. In the multiple-dose experiment, 2 groups of rats were dosed with 0.5 mg/kg body weight NMBA 3 times per week for 1 wk or 3 wk. The animals were sacrificed 24 h following the last treatment. Semiquantitative reverse-transcription polymerase chain reaction (RT-PCR) analysis demonstrated a reduction of CYP2A3 mRNA expression in lung and esophagus from NMBA-treated animals compared to dimethyl sulfoxide (DMSO)-treated vehicle controls. This reduction in CYP2A3 mRNA was significant at 48 h in the esophagus and at 24 and 48 h in the lung following a single dose of 2 mg/kg body weight NMBA. In contrast, CYP2E1 mRNA expression remained unchanged in rat lung following NMBA treatment and no consistent pattern of expression could be observed in the esophagus. In the multiple-dose study, a 32% and 25% reduction in esophageal CYP2A3 mRNA expression was observed at 1 and 3 wk, respectively. Similar reductions in CYP2A3 mRNA expression were also observed in the lung. Further, esophageal explants derived from animals pretreated with NMBA in vivo demonstrated a reduced ability to metabolize the carcinogen in vitro as compared to explants from vehicle control animals. Taken together, these data provide further support for a potential role of CYP2A3 in NMBA metabolism in the rat esophagus. Data suggest that CYP2A3 levels in the rat esophagus can be a determinant of its ability to metabolize this carcinogen in vivo.

Piroxicam is an ineffective inhibitor of N-nitrosomethylbenzylamine-induced tumorigenesis in the rat esophagus.

Epidemiological studies indicate an association between the frequent use of nonsteroidal anti-inflammatory drugs and decreased risk for esophageal cancer. These studies suggest that limiting excess prostaglandin production, via inhibition of cyclooxygenase (COX)-mediated arachidonic acid metabolism, may be an important strategy for the prevention of this type of malignancy. N-Nitrosomethylbenzylamine (NMBA)-induced tumorigenesis in the rat esophagus is a model of human esophageal squamous cell carcinoma used for investigations of chemical carcinogenesis and for the evaluation of putative chemopreventive agents. In this study, we characterized COX-mediated arachidonic acid metabolism in NMBA-induced rat esophageal tumorigenesis by measuring COX-1 and COX-2 expression and prostaglandin E(2) production. In addition, we evaluated the ability of piroxicam, a potent COX inhibitor, to prevent postinitiation events of NMBA-induced tumorigenesis in the rat esophagus. After a 2-week acclimatization period, groups of 30 male F344 rats received s.c. injections of NMBA (0.5 mg/kg b.w.) three times/week for 5 weeks. Seventy-two h after the final NMBA treatment and for the remainder of the study, piroxicam was administered in the diet at 200 and 400 ppm. Twenty-five weeks after the initiation of NMBA treatment, we observed an elevation in COX mRNA and protein expression and prostaglandin E(2) production in NMBA-treated esophageal tissues compared with normal epithelium. However, these changes were associated with data indicating that a COX inhibitor is not preventive in NMBA-induced rat esophageal tumorigenesis. Administration of piroxicam in the diet produced no significant reductions in esophageal tumor incidence, multiplicity, or size. The reasons for the lack of effect are largely unknown but may be related to the inability of piroxicam to modulate other biochemical pathways involved in NMBA-induced tumorigenesis.

Dynamic and biphasic modulation of nitrosation reaction by superoxide dismutases.

It has been shown that superoxide dismutase (SOD) can both potentiate and attenuate NO-mediated toxicity. This present study investigated the role of SOD and GSH in a sustained nitrosative and oxidative environment simulated by the nitric oxide (NO) and superoxide (O(2)(.-)) donor, 3-morpholinosydnonimine (SIN-1). We describe, for the first time, that SOD modulates nitrosative chemistry in a dynamic fashion that is both concentration and time-dependent. Specifically, our results show that SOD's effects on nitrosation are biphasic in nature i.e., while lower concentrations of SOD are pronitrosative, higher SOD concentrations inhibit nitrosation. However, even those initially inhibitory higher SOD concentrations became pronitrosative over time. In the presence of physiologically relevant levels of GSH, SOD predominantly exhibits a pronitrosative effect, with a complete loss of antinitrosative effects noted at higher levels of GSH. Our findings likely reflect the complex and dynamic nature of SOD interactions with oxidative and nitrosative species.

Motion segment stiffness measured without physiological levels of axial compressive preload underestimates the in vivo values in all six degrees of freedom.

UNLABELLED: Axial preload is known to alter the mechanical properties of spinal motion segments. The objective of this study was to compare the experimentally measured load-displacement behavior (stiffness, hysteresis and linearity) of porcine lumbar motion segments in vitro with physiological axial compressive preloads of 0, 200 and 400 N equilibrated in a physiological fluid environment. At each preload, displacements in each of six degrees of freedom (+/- 0.3 mm AP and lateral translations, +/- 0.2 mm axial translation, +/- 1 degrees lateral bending and +/- 0.8 degrees flexion/extension and torsional rotations) were imposed. The resulting forces and moments were recorded. Tests were repeated after removal of posterior elements. Using least squares, the forces at the vertebral body center were related to the displacements by a symmetric 6x6 stiffness matrix. The stiffness, hysteresis area and linearity of six diagonal and two off-diagonal load-displacement relationships were examined for differences between preload conditions. RESULTS: Mean values of the diagonal terms of the stiffness matrix for intact porcine motion segments increased significantly by an average factor of 2.2 and 2.9 with 200 and 400 N axial compression respectively (p<0.001). Increases for isolated disc specimens averaged 4.6 and 6.9 times with 200 and 400 N preload (p<0.001). Changes in hysteresis correlated with the changes in stiffness. The load-displacement relationships were progressively more linear with increasing preload (R2 = 0.82, 0.97 and 0.98 at 0, 200 and 400 N axial compression respectively). Motion segment and disc load-displacement behaviors were stiffer, more linear and had greater hysteresis with axial compressive preloads.