More than half of the reported leukoplakia lesions with biopsies showed ND. However, even lesions characterized as ND were associated with an increased risk of development of squamous cell carcinoma. Importantly, the risk of developing malignancy appears to correlate with the severity of dysplasia present on initial biopsy. Because clinical examination does not accurately predict the risk of malignancy, future studies, including genomic evaluation of this lesion, may be necessary to further characterize its biologic behavior.

Oxidative stress results when the balance between the production of reactive oxygen species (ROS) overrides the antioxidant capability of the target cell; oxidative damage from the interaction of reactive oxygen with critical cellular macromolecules may occur. ROS may interact with and modify cellular protein, lipid, and DNA, which results in altered target cell function. The accumulation of oxidative damage has been implicated in both acute and chronic cell injury including possible participation in the formation of cancer. Acute oxidative injury may produce selective cell death and a compensatory increase in cell proliferation. This stimulus may result in the formation of newly initiated preneoplastic cells and/or enhance the selective clonal expansion of latent initiated preneoplastic cells. Similarly, sublethal acute oxidative injury may produce unrepaired DNA damage and result in the formation of new mutations and, potentially, new initiated cells. In contrast, sustained chronic oxidative injury may lead to a nonlethal modification of normal cellular growth control mechanisms. Cellular oxidative stress can modify intercellular communication, protein kinase activity, membrane structure and function, and gene expression, and result in modulation of cell growth. We examined the role of oxidative stress as a possible mechanism by which nongenotoxic carcinogens may function. In studies with the selective mouse liver carcinogen dieldrin, a species-specific and dose-dependent decrease in liver antioxidant concentrations with a concomitant increase in ROS formation and oxidative damage was seen. This increase in oxidative stress correlated with an increase in hepatocyte DNA synthesis. Antioxidant supplementation prevented the dieldrin-induced cellular changes. Our findings suggest that the effect of nongenotoxic carcinogens (if they function through oxidative mechanisms) may be amplified in rodents but not in primates because of rodents' greater sensitivity to ROS. These results and findings reported by others support a potential role for oxidative-induced injury in the cancer process specifically during the promotion stage.ImagesFigure 5Figure 6Figure 9

Rotenone inhibits spontaneously and chemically induced hepatic tumorigenesis in rodents through the induction of apoptosis. However, the mechanism for the induction of apoptosis by rotenone has not been defined. Mitochondrial dysfunction, in particular the induction of the mitochondrial membrane permeability transition (MPT), has been implicated in the cascade of events involved in the induction of apoptosis. Inhibition of the mitochondrial electron-transport chain reduces the mitochondrial transmembrane potential (delta(psi)m), which may induce the formation of the mitochondrial permeability transition pore and the subsequent MPT. Fluorescent microscopy of Hoechst 33258-stained WB-F344 cells, a rat-liver cell line, was utilized to examine the effect of the mitochondrial respiratory chain inhibitor, rotenone (0.5-5 microM), atractyloside (5-10 microM), and cyclosporin A (2.5-10 microM) on apoptosis. A time- and concentration-dependent increase in liver cell apoptosis was observed following treatment with rotenone and atractyloside (11.7- and 7.7-fold, respectively, over solvent control). Cotreatment with 7.5- and 10 microM-cyclosporin A for 12 h inhibited the apoptogenicity of 5-microM rotenone treatment. A similar effect was observed following cyclosporin A cotreatment with atractyloside. Rotenone induced a rapid increase in apoptosis (within 20 min of treatment). By 2 h of treatment, the morphological appearance of apoptosis was similar to that observed in cultures treated continuously with rotenone for 12 h. Inhibition studies demonstrated that cyclosporin A prevented apoptosis if the exposure to it occurred prior to the 20-min threshold necessary to induce apoptosis by rotenone. Mitochondrial function was examined by staining with the mitochondrial membrane potential (delta(psi)m)-sensitive fluorochrome, MitoTracker Red (CMXRos) and confirmed utilizing cytofluorometric analysis of DiOC6(3)-stained cells. Rotenone (5.0-microM) and atractyloside (5.0-microM) reduced the percent of CMXRos or DiOC6(3)-positive (delta(psi)m-positive) liver cells within 15 min and throughout the duration of the study (6 h) to approximately 65-80% and 50-80% of control. However, cotreatment with concentrations of cyclosporin A that inhibited the apoptogenicity of rotenone and atractyloside prevented the rotenone- and atractyloside-induced reduction of the delta(psi)m. Therefore, the apoptogenic effect of rotenone and atractyloside appears to occur rapidly (within 20 min) and is irreversible once mitochondrial damage occurs. The inhibition of the rotenone- and atractyloside-induced apoptosis and mitochondrial dysfunction by cyclosporin A suggests the MPT may be involved in the induction of apoptosis by rotenone.

A number of chemical mediators can induce human keratinocytes and epidermal-derived carcinomas to undergo apoptosis, or programmed cell death. Recent evidence suggests pro-inflammatory cytokines, such as interleukin-1␤ or transforming growth factor ␣, protects carcinomas from numerous pro-apoptotic stimuli. Platelet-activating factor (1-alkyl-2-acetyl-3-glycerophosphocholine; PAF) is a lipid mediator with pro-inflammatory effects on numerous cell types. Although PAF can be metabolized to other bioactive lipids, the majority of PAF effects occur through activation of a G proteincoupled receptor. Using a model system created by retroviral transduction of the PAF receptor (PAF-R) into the PAF-R-negative human epidermal cell line KB and the PAF-R-expressing keratinocyte cell line HaCaT, we now demonstrate that activation of the epidermal PAF-R results in protection from apoptosis induced by tumor necrosis factor (TNF) ␣ or TNF-related apoptosisinducing ligand. The PAF-mediated protection was inhibited by PAF-R antagonists, and protection did not occur in PAF-R-negative KB cells. Additionally, we show protection from TNF␣-or TRAIL-induced apoptosis by PAF-R activation is dependent on the transcription factor nuclear factor (NF)-B, because PAF-R activationinduced NF-B and epidermal cells transduced with a super-repressor form of inhibitor B were not protected by the PAF-R. These studies provide a mechanism whereby the epidermal PAF-R, and possibly other G protein-coupled receptors, can exert anti-apoptotic effects through an NF-B-dependent process.

The present study evaluated the effect of di-2-ethylhexyl phthalate (DEHP) on gap-junctional intercellular communication (GJIC), peroxisomal beta-oxidation (PBOX) activity, and replicative DNA synthesis in several rodent species with differing susceptibilities to peroxisome proliferator-induced hepatic tumorigenesis. A low (non-tumorigenic) and high (tumorigenic) dietary concentration of DEHP was administered to male F344 rats for 1, 2, 4, and 6 weeks. Additionally, a previously non-tumorigenic dose (1000 ppm) and tumorigenic dose of DEHP (12,000 ppm), as determined by chronic bioassay data, were examined following 2 weeks dietary administration. Male B6C3F1 mice were fed the non-tumorigenic concentration, 500 ppm, and the tumorigenic concentration, 6000 ppm, of DEHP for two and four weeks. The hepatic effects of low and high concentrations of DEHP, 1000 and 6000 ppm, were also examined in male Syrian Golden hamsters (refractory to peroxisome proliferator-induced tumorigenicity). In rat and mouse liver, a concentration-dependent increase in the relative liver weight, PBOX activity, and replicative DNA synthesis was observed at the earliest time point examined. Concurrent to these observations was an inhibition of GJIC. In hamster liver, a slight increase in the relative liver weight, PBOX activity, and replicative DNA synthesis was observed. However, these effects were not of the same magnitude or consistency as those observed in rats or mice. Furthermore, DEHP had no effect on GJIC in hamster liver at any of the time points examined (2 and 4 weeks). HPLC analysis of DEHP and its primary metabolites, mono-2-ethylhexyl phthalate (MEHP), and phthalate acid (PA), indicated a time- and concentration-dependent increase in the hepatic concentration of MEHP. At equivalent dietary concentrations and time points, the presence of MEHP, the primary metabolite responsible for the hepatic effects of DEHP, demonstrated a species-specific response. The largest increase in the hepatic concentration of MEHP was observed in mice, which was greater than the concentration observed in rats. The hepatic concentration of MEHP was lowest in hamsters. Hepatic concentrations of DEHP and phthalic acid were minimal and did not correlate with concentration and time. Collectively, these data demonstrate the inhibition of hepatic GJIC and increased replicative DNA synthesis correlated with the observed dose- and species-specific tumorigenicity of DEHP and may be predictive indicators of the nongenotoxic carcinogenic potential of phthalate esters.

Several phthalate esters, compounds used as plasticizers in a variety of commercial products, have been shown to induce hepatic tumors in rodents. In this study, the comparative effects of phthalate monoesters on inhibition of gap junctional intercellular communication and induction of peroxisomal beta-oxidation were assessed in primary cultured hepatocytes from rats, mice, hamsters, cynomolgus monkeys, and humans. A human liver cell line was also utilized. Eight monoesters examined included mono-2-ethylhexyl phthalate (MEHP), mono-n-octyl phthalate (MNOP), mono-isononyl phthalate (MINP, 3 types, -1, -2, and -3), mono-isoheptyl phthalate (MIHP), mono-isodecyl phthalate (MIDP), and mono-(heptyl, nonyl, undecyl) phthalate (M711P). Gap junctional intercellular communication was measured 4 and 24 h after treatment by lucifer yellow dye coupling. Gap junctional intercellular communication was inhibited in rat and mouse hepatocytes by all eight monoesters in a concentration-dependent manner. In most cases, gap junctional intercellular communication was significantly reduced at the lowest concentrations tested (50 pM). Inhibition of gap junctional intercellular communication in rodent cells was substantially reversed within 24 h of monoester removal. In contrast, cell-to-cell communication was not inhibited in hamster, cynomolgus, or human hepatocytes or in a human liver cell line at any concentration examined. In rat hepatocytes, peroxisomal beta-oxidation was elevated after treatment with MEHP, MINP, MIHP, and MIDP but not MNOP or M711P, and with all but MIHP in mouse hepatocytes. The eight phthalates produced no marked change on peroxisomal beta-oxidation in hepatocytes from other species. These data provide additional evidence that the toxicological effects of phthalate esters are species specific.

Objectives/Hypothesis Malignant transformation of laryngeal keratosis has been reported in a substantial subset of patients, yet reliable criteria for predicting patients most at risk have yet to be determined. Current methods for determining dysplasia ratings are susceptible to errors in biopsy sampling and interpretation. An understanding of the genetic underpinnings of the progression of vocal fold tumorigenesis may contribute to the creation of reliable and predictive diagnostic criteria. We hypothesized that genetic expression markers distinguish patients with keratotic noncancerous vocal fold lesions from invasive carcinoma. Study Design Observational cross-sectional study. Methods Real-time polymerase chain reaction (RT-PCR) was used to compare expression of 84 cancer pathway genes of patients following histologic diagnosis of nondysplastic keratotic epithelium (ND) (n = 7), dysplastic keratotic epithelium (DYS) (n = 3), and invasive carcinoma (CA) (n = 7). All patients had a clinical diagnosis of leukoplakia, and biopsies were obtained from true vocal fold tissue. Results Four genes (IGF-1, EPDR1, MMP-2, S100A4) were significantly upregulated in DYS over the ND group. Seven genes were significantly upregulated in CA over the DYS group, and 31 genes were significantly upregulated in CA over the ND group (P < .02). The expression of matrix metalloproteinases (MMP-1, MMP-2, MMP-9) was found to statistically differentiate the groups (P < .02) and suggested disease progression associated with extracellular matrix degradation and angiogenesis promotion. Conclusions With these preliminary array data, we demonstrate the feasibility of using RT-PCR to identify distinct genetic expression between diagnostic groups. Characterization of genetic changes marking the progression of vocal fold tumorigenesis may lead to robust diagnostic criteria in the future.

The tumor promotion stage of chemical carcinogenesis has been shown to exhibit a persistence of cellular effects during treatment and the reversibility of these changes upon cessation of treatment. Inhibition of gap-junctional intercellular communication and increased replicative DNA synthesis appear to be important in this process. The present study assessed the persistence and reversibility of gap-junctional intercellular communication inhibition, peroxisomal proliferation, and replicative DNA synthesis in livers from male F344 rats and B6C3F1 mice. Dietary administration of 20,000 mg/kg DEHP to male rats for 2 weeks decreased intercellular communication (67% of control) and enhanced replicative DNA synthesis (4.8-fold over control). Elevation of the relative liver weight and the induction of peroxisomal beta oxidation were also observed following treatment with 20,000 mg/Kg DEHP for 2 weeks. Following DEHP administration at a dose of 6000 mg/kg for 18 months, inhibition of gap-junctional intercellular communication persisted, and the relative liver weight and induction of peroxisomal beta oxidation remained elevated in both rats and male B6C3F1 mice. Treatment of rats and mice with phenobarbital for 18 months (500-mg/kg diet) also produced an increase in relative liver weight and a decrease in cell-to-cell communication. In recovery studies in which DEHP was administered to male F344 rats for 2 weeks and then withdrawn, the relative liver weight, rate of peroxisomal beta oxidation, increase in replicative DNA synthesis, and inhibition of gap-junctional intercellular communication returned to control values within 2 to 4 weeks after DEHP treatment ceased. Recovery studies with phenobarbital produced similar results. The primary active metabolite of DEHP, mono-2-ethylhexyl phthalate (MEHP), was detected in the livers of animals treated with DEHP for greater than 2 weeks. However, it could not be detected after removal of DEHP from the diet for 2 weeks. This study demonstrated that inhibition of gap-junctional intercellular communication, along with indicators of peroxisomal proliferation, including increased relative liver weight and enhanced peroxisomal beta oxidation, persist while DEHP treatment continues but reverses when treatment is stopped. Studies with phenobarbital produced a similar pattern of response.