Chemopreventive effects of a selective nitric oxide synthase inhibitor on carcinogen-induced rat esophageal tumorigenesis

The inducible nitric oxide synthase (iNOS) generates a high concentration of nitric oxide (NO) in tissues. Increased NO production is associated with many disorders including esophageal cancer. Previous studies in our laboratory demonstrated an association between increased iNOS expression and the development of N-nitrosomethylbenzylamine (NMBA)-induced tumors in the rat esophagus. On the basis of these observations, we initiated a bioassay to evaluate the ability of S,S'-1,4-phenylene-bis(1,2-ethanediyl)bis-isothiourea (PBIT), a selective iNOS inhibitor, to prevent the progression of esophageal tumors in rats preinitiated with NMBA. Rats were given s.c. injections of NMBA (0.25 mg/kg body weight) three times per week for 5 weeks. One week later, they were fed a synthetic diet containing either 50 or 100 ppm PBIT until the end of the bioassay (25 weeks). PBIT reduced the incidence of esophageal cancer from 96% in NMBA-treated rats to 83% and 77% (P < 0.05) in rats treated with 50 and 100 ppm PBIT, respectively. Tumor multiplicity was reduced from 3.64 +/- 0.42 tumors per esophagus in NMBA-treated rats to 1.79 +/- 0.25 (P < 0.001) and 1.50 +/- 0.24 (P < 0.0001) in rats treated with 50 and 100 ppm PBIT, respectively. PBIT reduced the production of NO in NMBA-induced preneoplastic and papillomatous esophageal lesions when compared with comparable lesions in rats treated with NMBA only. iNOS mRNA expression was not modulated by PBIT. These observations suggest that iNOS plays a role in tumor development and that its selective inhibitor, PBIT, significantly inhibits esophageal tumor progression presumably through reducing the production of NO.     

Integrated oral bioavailability projection using in vitro screening data as a selection tool in drug discovery

The objective of the analysis described herein is to examine the in vitro/in vivo relationship of estimated bioavailability values and also the applicability of the estimated in vitro bioavailability to lead candidate selection in drug discovery. To this end, in vitro ADME data from screening assays as well as in vivo rat pharmacokinetic (PK) data were compiled for 140 compounds across therapeutic areas from the Pfizer library in Ann Arbor. The compounds span a broad range of structural types, including neutral, basic, and acidic compounds. Solubility and Caco-2 permeability data from in vitro ADME screening were used to calculate the fraction dose absorbed (FDp) using the physiologically based IDEA model. In vitro metabolic stability (t(1/2)) from human and rat liver microsomal incubations was converted to an in vitro intrinsic clearance value (CL(int)'), which was then scaled up to reflect in vivo clearance (CL) and hepatic extraction as described by Obach et al. [J. Pharmcol. Exp. Ther. 283 (1997) 46]. Subsequently, the in vitro/in vivo relationship between the measured bioavailability (F(obs)) in rats and the estimated bioavailability (F(est)) from FDp and predicted CL values was examined. The observed data suggest that compounds with low estimated in vitro bioavailability (F(est)<15%) are more likely to have low in vivo bioavailability (F(obs)<30%). Therefore, the present study indicates that in vitro estimation of bioavailability is an efficient tool to eliminate compounds having low bioavailability prior to in vivo characterization and therefore can be used to reduce attrition due to poor ADME properties in drug development.     

Attentional selection of superimposed surfaces cannot be explained by modulation of the gain of color channels

When two differently colored, superimposed patterns of dots rotate in opposite directions, this yields the percept of two superimposed transparent surfaces. If observers are cued to attend to one set of dots, they are impaired in making judgments about the other set. Since the two sets of dots are overlapping, the cueing effect cannot be explained by spatial attention. This has led to the interpretation that the impairment reflects surface-based attentional selection. However, recent single-unit recording studies in monkeys have found that attention can modulate the gain of neurons tuned for features such as color. Thus, rather than reflecting the selection of a surface, the behavioral effects might simply reflect a reduction in the gain of color channels selective for the color of the uncued set of dots (feature-based attention), as if viewing the surfaces through a colored filter. If so, then the impairment should be eliminated when the two surfaces are made the same color. Instead, we find that the impairment persists with no reduction in strength. Our findings thus rule out the color gain explanation.     

Exogenously cued attention triggers competitive selection of surfaces

It has been reported that when an endogenous cue directs attention to a brief translation of one of two superimposed surfaces, observers reliably report the direction of that translation as well as the direction of a second translation of the cued surface. In contrast, if the uncued surface translates second, direction judgments are severely impaired for several hundred milliseconds. We replicated this result, but found that the impairment survived the removal of the endogenous cue. The impairment is therefore not due to endogenously cued attention. Instead, a brief translation of one surface acts as an exogenous cue that triggers an automatic selection mechanism, which suppresses processing of the other surface. This study provides a clear case of exogenous cueing of surface-based attention. We relate these results to identified competitive selection mechanisms in visual cortex.     

Chemoprevention of esophageal tumorigenesis by dietary administration of lyophilized black raspberries

Fruit and vegetable consumption has consistently been associated with decreased risk of a number of aerodigestive tract cancers, including esophageal cancer. We have taken a "food-based" chemopreventive approach to evaluate the inhibitory potential of lyophilized black raspberries (LBRs) against N-nitrosomethylbenzylamine (NMBA)-induced esophageal tumorigenesis in the F344 rat, during initiation and postinitiation phases of carcinogenesis. Anti-initiation studies included a 30-week tumorigenicity bioassay, quantification of DNA adducts, and NMBA metabolism study. Feeding 5 and 10% LBRs, for 2 weeks prior to NMBA treatment (0.25 mg/kg, weekly for 15 weeks) and throughout a 30-week bioassay, significantly reduced tumor multiplicity (39 and 49%, respectively). In a short-term bioassay, 5 and 10% LBRs inhibited formation of the promutagenic adduct O(6)-methylguanine (O(6)-meGua) by 73 and 80%, respectively, after a single dose of NMBA at 0.25 mg/kg. Feeding 5% LBRs also significantly inhibited adduct formation (64%) after NMBA administration at 0.50 mg/kg. The postinitiation inhibitory potential of berries was evaluated in a second bioassay with sacrifices at 15, 25, and 35 weeks. Administration of LBRs began after NMBA treatment (0.25 mg/kg, three times per week for 5 weeks). LBRs inhibited tumor progression as evidenced by significant reductions in the formation of preneoplastic esophageal lesions, decreased tumor incidence and multiplicity, and reduced cellular proliferation. At 25 weeks, both 5 and 10% LBRs significantly reduced tumor incidence (54 and 46%, respectively), tumor multiplicity (62 and 43%, respectively), proliferation rates, and preneoplastic lesion development. Yet, at 35 weeks, only 5% LBRs significantly reduced tumor incidence and multiplicity, proliferation indices and preneoplastic lesion formation. In conclusion, dietary administration of LBRs inhibited events associated with both the initiation and promotion/progression stages of carcinogenesis, which is promising considering the limited number of chemopreventives with this potential.     

Post-initiation effects of chlorophyllin and indole-3-carbinol in rats given 1,2-dimethylhydrazine or 2-amino-3-methyl- imidazo

Chlorophyllin (CHL) is a water-soluble derivative of chlorophyll, the ubiquitous pigment in green and leafy vegetables, whereas indole-3-carbinol (I3C) is present in cruciferous vegetables such as cabbage, broccoli and cauliflower. In rats initiated with 1,2-dimethylhydrazine (DMH), CHL and I3C reportedly promoted or enhanced the incidence of colon tumors when they were administered after, or during and after the carcinogen exposure, respectively. The same compounds given post-initiation inhibited the formation of colonic aberrant crypts induced by heterocyclic amines, such as 2-amino-3-methylimidazo[4,5-f]quinoline (IQ), but tumor suppression was not examined in the latter studies. In the present investigation, male F344 rats were treated with IQ or DMH during the first 5 weeks of a 1 year study; IQ was given in the diet (0.03%), whereas DMH was administered once a week by s.c. injection (20 mg/kg body wt). Beginning 1 week after the last dose of IQ or DMH until sacrifice, rats received 0.001, 0.01 or 0.1% (w/v) CHL in the drinking water or 0.001, 0.01 or 0.1% I3C in the diet. Compared with controls given carcinogen alone, 0.1% I3C treatment suppressed the multiplicity of IQ-induced colon tumors, and CHL inhibited in a dose-related manner the incidence of IQ-induced liver tumors. However, 0.001% CHL increased significantly the multiplicity of DMH-induced colon tumors while having no effect on the colon tumors induced by IQ. These results indicate that both the choice of carcinogen as well as the dose of the tumor modulator can be important determinants of the events that occur during post-initiation exposure to CHL or I3C. Based on the present findings and data in the literature, it is possible for CHL and I3C to act as tumor promoters or anticarcinogens, depending upon the test species, initiating agent and exposure protocol.     

Assessment of adhesion assays for use with keratinocytes

Cell attachment, as a biological process, is an important aspect with respect to graft survival and "take". With the ever-increasing use of cultured epithelial autografts for coverage and re-epithelialization of wounds, the assessment of keratinocyte adhesion in vitro has become a more common requirement in studies involving extracellular matrix proteins and their receptor molecules. Cell adhesion has been well-documented in immunological research, however keratinocyte adhesion has been investigated by manual counting (using methylene blue) or other less sensitive colorimetric methods. With the increase in number of fluorogenic probes available, their use as a sensitive alternative to radioactive labelling has been promoted in the literature. This study was carried out to investigate the possibility of using fluorescent probe 5,6-carboxyfluorescein diacetate succidimyl ester to achieve a more standardized assay in the assessment of keratinocyte adhesion. Adhesion was assessed on extracellular matrix proteins such as fibronectin, collagen types I & IV and laminin. We concluded that the fluorescent probe might provide greater sensitivity in measuring adhesion, however it may be cytotoxic to keratinocytes. Pre-labelling of keratinocytes may affect cellular functions such as adhesion and even proliferation and consequently the probe must be chosen with care.     

Emergency department screening for asymptomatic sexually transmitted infections

OBJECTIVES: This study assessed the prevalence and correlates of asymptomatic genital tract infection with Neisseria gonorrhoeae and Chlamydia trachomatis among emergency department patients. METHODS: Individuals seeking emergency department evaluation for nongenitourinary complaints provided urine samples for N gonorrhoeae and C trachomatis testing by ligase chain reaction and completed a sociodemographic and behavioral questionnaire. RESULTS: Asymptomatic N gonorrhoeae or C trachomatis was found in 9.7% of persons tested. Correlates of C trachomatis infection included younger age, residence in high-morbidity zip code areas, previous history of N gonorrhoeae or C trachomatis, and number of sex partners in the past year. CONCLUSIONS: Urine-based screening of asymptomatic emergency department patients detected significant numbers of N gonorrhoeae and C trachomatis infections. Targeted screening programs may contribute to community-level prevention and control of sexually transmitted infections.     

Incidence and effects of Ha-ras codon 12 G-->A transition mutations in preneoplastic lesions induced by N-nitrosomethylbenzylamine in the rat esophagus

N-nitrosomethylbenzylamine (NMBA)-induced rat esophageal tumorigenesis is an important model for squamous cell carcinoma of the human esophagus. In this model, previous studies have shown that the GGA-->GAA Ha-ras codon 12 mutation is present in the majority of papillomas. No other Ha-ras mutation has been identified. Studies using other models of chemical carcinogenesis suggest that Ha-ras activation has a critical role during tumor initiation. We have used laser-capture microdissection and polymerase chain reaction-restriction fragment length polymorphism analysis to study the role of codon 12 Ha-ras mutation at various stages of tumor development in the rat esophagus. Our results indicate that Ha-ras mutation was present infrequently (4.3%) in premalignant lesions. The incidence of Ha-ras mutation was high in papillomas (57.1%), however, and 50% of papillomas expressed mutant Ha-ras RNA message. Additionally, there was a linear trend correlating increased incidence of Ha-ras mutation with later papilloma stage. These data suggest the role of ras activation later in neoplastic development. To evaluate the potential mechanism of action by which Ha-ras contributes to promotion and progression in this model, we compared mRNA expression of cyclin D1 and p27 in Ha-ras mutant and Ha-ras normal papillomas. We found no differences in mRNA expression of either cyclin D1 or p27 between these two papilloma populations. Our data suggest an important paradigm shift for the role of ras mutations in this model of chemical carcinogenesis, indicating a functional role of Ha-ras activation in promotion/progression and not in the initiation phase of NMBA-induced papillomagenesis.