Perception of higher-order affordances for kicking in soccer.

We investigated the perception of higher-order interpersonal affordances for kicking that emerged from lower-order personal and interpersonal affordances in the context of soccer. Youth soccer players reported the minimum gap width between two confederates through which they could kick a ball. In Experiment 1, we independently manipulated the egocentric distance of gaps from participants, and the nominal role of the confederates, either as teammates or opponents. In Experiment 2, we additionally varied the direction in which the confederates were facing, either together (i.e., into the gap) or away (i.e., away from the gap). Perceived minimum kickable gap width was larger for farther egocentric distances, when confederates were identified as opponents rather than as teammates, and (in Experiment 2) when confederates faced toward, rather than away from the gap. In both experiments, these main effects were subsumed in statistically significant interactions. We argue that these interactions reveal perception of higher-order interpersonal affordances for kicking that emerged from the simultaneous influence of lower-order affordances. The results are compatible with the hypothesis that these higher-order affordances were perceived, as such, and were not additively combined from independent perception of underlying, lower-order affordances. (PsycInfo Database Record (c) 2023 APA, all rights reserved).

MicroRNA profiling of carcinogen-induced rat colon tumors and the influence of dietary spinach.

SCOPE: MicroRNA (miRNA) profiles are altered in chronic conditions such as cardiovascular disease, diabetes, neurological disorders, and cancer. A systems biology approach was used to examine, for the first time, miRNAs altered in rat colon tumors induced by 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine (PhIP), a heterocyclic amine carcinogen from cooked meat. METHODS AND RESULTS: Among the most highly dysregulated miRNAs were those belonging to the let-7 family. Subsequent computational modeling and target validation identified c-Myc and miRNA-binding proteins Lin28A/Lin28B (Lin28) as key players, along with Sox2, Nanog, and Oct-3/4. These targets of altered miRNAs in colon cancers have been implicated in tumor recurrence and reduced patient survival, in addition to their role as pluripotency factors. In parallel with these findings, the tumor-suppressive effects of dietary spinach given postinitiation correlated with elevated levels of let-7 family members and partial normalization of c-myc, Sox2, Nanog, Oct-3/4, HmgA2, Dnmt3b, and P53 expression. CONCLUSION: We conclude that the let-7/c-Myc/Lin28 axis is dysregulated in heterocyclic amine-induced colon carcinogenesis, and that the tumor suppressive effects of dietary spinach are associated with partial normalization of this pathway.

Cancer chemoprevention by dietary chlorophylls: a 12,000-animal dose-dose matrix biomarker and tumor study.

Recent pilot studies found natural chlorophyll (Chl) to inhibit carcinogen uptake and tumorigenesis in rodent and fish models, and to alter uptake and biodistribution of trace (14)C-aflatoxin B1 in human volunteers. The present study extends these promising findings, using a dose-dose matrix design to examine Chl-mediated effects on dibenzo(def,p)chrysene (DBC)-induced DNA adduct formation, tumor incidence, tumor multiplicity, and changes in gene regulation in the trout. The dose-dose matrix design employed an initial 12,360 rainbow trout, which were treated with 0-4000ppm dietary Chl along with 0-225ppm DBC for up to 4weeks. Dietary DBC was found to induce dose-responsive changes in gene expression that were abolished by Chl co-treatment, whereas Chl alone had no effect on the same genes. Chl co-treatment provided a dose-responsive reduction in total DBC-DNA adducts without altering relative adduct intensities along the chromatographic profile. In animals receiving DBC alone, liver tumor incidence (as logit) and tumor multiplicity were linear in DBC dose (as log) up to their maximum-effect dose, and declined thereafter. Chl co-treatment substantially inhibited incidence and multiplicity at DBC doses up to their maximum-effect dose. These results show that Chl concentrations encountered in Chl-rich green vegetables can provide substantial cancer chemoprotection, and suggest that they do so by reducing carcinogen bioavailability. However, at DBC doses above the optima, Chl co-treatments failed to inhibit tumor incidence and significantly enhanced multiplicity. This finding questions the human relevance of chemoprevention studies carried out at high carcinogen doses that are not proven to lie within a linear, or at least monotonic, endpoint dose-response range.

E2F4 and ribonucleotide reductase mediate S-phase arrest in colon cancer cells treated with chlorophyllin.

Chlorophyllin (CHL) is a water-soluble derivative of chlorophyll that exhibits cancer chemopreventive properties, but which also has been studied for its possible cancer therapeutic effects. We report here that human colon cancer cells treated with CHL accumulate in S-phase of the cell cycle, and this is associated with reduced expression levels of p53, p21, and other G(1)/S checkpoint controls. At the same time, E2F1 and E2F4 transcription factors become elevated and exhibit increased DNA binding activity. In CHL-treated colon cancer cells, bromodeoxyuridine pulse-chase experiments provided evidence for the inhibition of DNA synthesis. Ribonucleotide reductase (RR), a pivotal enzyme for DNA synthesis and repair, was reduced at the mRNA and protein level after CHL treatment, and the enzymatic activity was inhibited in a concentration-dependent manner both in vitro and in vivo. Immunoblotting revealed that expression levels of RR subunits R1, R2, and p53R2 were reduced by CHL treatment in HCT116 (p53(+/+)) and HCT116 (p53(-/-)) cells, supporting a p53-independent mechanism. Prior studies have shown that reduced levels of RR small subunits can increase the sensitivity of colon cancer cells to clinically used DNA-damaging agents and RR inhibitors. We conclude that by inhibiting R1, R2, and p53R2, CHL has the potential to be effective in the clinical setting, when used alone or in combination with currently available cancer therapeutic agents.

Nonlinear cancer response at ultralow dose: a 40800-animal ED(001) tumor and biomarker study.

Assessment of human cancer risk from animal carcinogen studies is severely limited by inadequate experimental data at environmentally relevant exposures and by procedures requiring modeled extrapolations many orders of magnitude below observable data. We used rainbow trout, an animal model well-suited to ultralow-dose carcinogenesis research, to explore dose-response down to a targeted 10 excess liver tumors per 10000 animals (ED(001)). A total of 40800 trout were fed 0-225 ppm dibenzo[a,l]pyrene (DBP) for 4 weeks, sampled for biomarker analyses, and returned to control diet for 9 months prior to gross and histologic examination. Suspect tumors were confirmed by pathology, and resulting incidences were modeled and compared to the default EPA LED(10) linear extrapolation method. The study provided observed incidence data down to two above-background liver tumors per 10000 animals at the lowest dose (that is, an unmodeled ED(0002) measurement). Among nine statistical models explored, three were determined to fit the liver data well-linear probit, quadratic logit, and Ryzin-Rai. None of these fitted models is compatible with the LED(10) default assumption, and all fell increasingly below the default extrapolation with decreasing DBP dose. Low-dose tumor response was also not predictable from hepatic DBP-DNA adduct biomarkers, which accumulated as a power function of dose (adducts = 100 x DBP(1.31)). Two-order extrapolations below the modeled tumor data predicted DBP doses producing one excess cancer per million individuals (ED(10)(-6)) that were 500-1500-fold higher than that predicted by the five-order LED(10) extrapolation. These results are considered specific to the animal model, carcinogen, and protocol used. They provide the first experimental estimation in any model of the degree of conservatism that may exist for the EPA default linear assumption for a genotoxic carcinogen.

Rainbow trout (Oncorhynchus mykiss) and ultra-low dose cancer studies.

Cancer risk assessment utilizing rodents requires extrapolation across five orders of magnitude to estimate the Virtually Safe Dose (VSD). Regulatory agencies rely upon the Linear Extrapolated Dose (LED) except when sufficient information on mechanism of action justifies alternative models. Rainbow trout (Oncorhynchus mykiss) has been utilized at Oregon State University as a model for human cancer for forty years. Low cost and high capacity, made possible by our unique facility, along with low spontaneous background and high sensitivity, allow design and conduct of statistically challenging studies not possible in rodents. Utilization of custom microarrays demonstrates similarities in gene expression in trout and human hepatocellular carcinoma (HCC). We have completed one study employing over 42,000 trout with dibenzo[a,l]pyrene (DBP) and determined the dose resulting in 1 additional cancer in 5000 animals, a 50-fold enhancement over the mouse ED(01) study. Liver tumor incidence at low dose deviated significantly from linearity (concave down), whereas, DBP-DNA adductions deviated slightly (convex up). A second study is underway with aflatoxin B(1) (AFB(1)). Results to date indicate AFB(1) at low dose, in contrast to DBP, elicits a linear dose-response function on the log-log scale which falls below the LED with a slope slightly greater than 1.0. Such studies demonstrate the statistical power of the trout cancer model and strengthen the case for incorporation of these data-sets into risk assessment for these environmental human carcinogens.

Identifying efficacious approaches to chemoprevention with chlorophyllin, purified chlorophylls and freeze-dried spinach in a mouse model of transplacental carcinogenesis.

The carcinogenic potential of dibenzo[a,l]pyrene (DBP) has been well characterized in numerous animal models. We have previously documented that a single dose of 15 mg/Kg DBP to pregnant mice late in gestation (GD 17) produces an aggressive T-cell lymphoma as well as lung and liver cancer in offspring. The current study examines the chemopreventative properties of chlorophyllin (CHL) and chlorophyll (Chl) in this transplacental carcinogenesis model. Pregnant B6129SF1 females, bred to 129S1/SvIm males, received purified diets incorporated with either 2000 p.p.m. CHL, 2000 p.p.m. Chl or 10% freeze-dried spinach beginning at gestation day 9. Lymphoma-dependent mortality was not significantly altered by maternal consumption of any of the diet and little effect on lung tumor burden in mice surviving to 10 months of age was observed. However, coadministration of CHL at 380 mg/Kg with DBP by gavage (molar ratio of 10:1, CHL:DBP) provided significant protection against DBP-initiated carcinogenesis. Offspring born to dams receiving CHL co-gavaged with DBP exhibited markedly less lymphoma-dependent mortality (P < 0.001). The degree of protection by CHL, compared with controls dosed with DBP in tricaprylin (TCP) as the vehicle, was less marked, but still significant. Coadministration of CHL (TCP as vehicle) also reduced lung tumor multiplicity in mice by approximately 50% and this was observed throughout the study (P < 0.005). This is the first demonstration that CHL can provide potent chemoprotection in a transplacental carcinogenesis model and support a mechanism involving complex-mediated reduction of carcinogen uptake.

Protective versus promotional effects of white tea and caffeine on PhIP-induced tumorigenesis and beta-catenin expression in the rat.

A 1 year carcinogenicity bioassay was conducted in rats treated with three short cycles of 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine (PhIP)/high-fat (HF) diet, followed by 2% white tea (wt/vol), 0.05% epigallocatechin-3-gallate (EGCG) or 0.065% caffeine as sole source of fluid intake. Thirty-two percent of the PhIP/HF controls survived to 1 year, compared with 50, 48.7 and 18.2% in groups given white tea, EGCG and caffeine, respectively. After 1 year, PhIP/HF controls had tumors in the colon, skin, small intestine, Zymbal's gland, salivary gland and pancreas. For all sites combined, excluding the colon, tumor incidence data were as follows: PhIP/HF 69.5%, PhIP/HF + EGCG 48.7%, PhIP/HF + white tea 46.9% and PhIP/HF + caffeine 13.3%. Unexpectedly, a higher incidence of colon tumors was detected in rats post-treated with white tea (69%) and caffeine (73%) compared with the 42% incidence in PhIP/HF controls. In the colon tumors, beta-catenin mutations were detected at a higher frequency after caffeine posttreatment, and there was a shift toward more tumors harboring substitutions of Gly34 with correspondingly high protein and messenger RNA expression seen for both beta-catenin and c-Myc. c-Myc expression exhibited concordance with tumor promotion, and there was a concomitant increase in cell proliferation versus apoptosis in colonic crypts. A prior report described suppression of PhIP-induced colonic aberrant crypts by the same test agents, but did not incorporate a HF diet. These findings are discussed in the context of epidemiological data which do not support an adverse effect of tea and coffee on colon tumor outcome-indeed, some such studies suggest a protective role for caffeinated beverages.

Low-dose dietary chlorophyll inhibits multi-organ carcinogenesis in the rainbow trout.

We recently reported that chlorophyll (Chl) strongly inhibits aflatoxin B(1) preneoplasia biomarkers in rats when administered by co-gavage (Simonich et al., 2007. Natural chlorophyll inhibits aflatoxin B1-induced multi-organ carcinogenesis in the rat. Carcinogenesis 28, 1294-1302.). The present study extends this by examining the effects of dietary Chl on tumor development, using rainbow trout to explore ubiquity of mechanism. Duplicate groups of 140 trout were fed diet containing 224 ppm dibenzo[a,l]pyrene (DBP) alone, or with 1000-6000 ppm Chl, for 4 weeks. DBP induced high tumor incidences in liver (51%) and stomach (56%), whereas Chl co-fed at 2000, 4000 or 6000 ppm reduced incidences in stomach (to 29%, 23% and 19%, resp., P<0.005) and liver (to 21%, 28% and 26%, resp., P<0.0005). Chlorophyllin (CHL) at 2000 ppm gave similar protection. Chl complexed with DBP in vitro (2Chl:DBP, K(d1)=4.44+/-0.46 microM, K(d2)=3.30+/-0.18 microM), as did CHL (K(d1)=1.38+/-0.32 microM, K(d2)=1.17+/-0.05 microM), possibly explaining their ability to inhibit DBP uptake into the liver by 61-63% (P<0.001). This is the first demonstration that dietary Chl can reduce tumorigenesis in any whole animal model, and that it may do so by a simple, species-independent mechanism.

Protective effect of dietary tomatine against dibenzo[a,l]pyrene (DBP)-induced liver and stomach tumors in rainbow trout.

The potential anti-carcinogenic effects of tomatine, a mixture of commercial tomato glycoalkaloids alpha-tomatine and dehydrotomatine (10:1), were examined in the rainbow trout chemoprevention model. Prior to the chemoprevention study, a preliminary toxicity study revealed that tomatine in the diet fed daily at doses from 100 to 2000 parts per million (ppm) for 4 weeks was not toxic to trout. For the tumor study, replicate groups of 105 trout were fed diets containing dibenzo[a,l]pyrene (DBP) alone (224 ppm), (N = 3), DBP plus tomatine at 2000 ppm (N = 2), tomatine alone (N = 2), or control diet (N = 2) for 4 weeks. The fish were then returned to control diet for 8 months and necropsied for histopathology. Dietary tomatine was found to reduce DBP-initiated liver tumor incidence from 37.0 to 19.0% and stomach tumor incidence from 46.4 to 29.4%. Tomatine also reduced stomach tumor multiplicity. The tomatine-containing diets did not induce mortality, change in fish weights, or liver weights. No adverse pathological effects in the tissues of the fish on the tomatine diets were observed. Dose-response and chemopreventive mechanisms for tomatine protection remain to be examined. This is the first report on the anticarcinogenic effects of tomatine in vivo.

Comparison of white tea, green tea, epigallocatechin-3-gallate, and caffeine as inhibitors of PhIP-induced colonic aberrant crypts.

There is growing interest in the possible health benefits of tea. We reported previously on the inhibition by white tea of 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine (PhIP)-induced colonic aberrant crypt foci (ACF) in the rat (4). To distinguish between blocking and suppressing effects, and thus provide mechanistic insights into prevention during the initiation versus post-initiation phases of carcinogenesis, white tea, and green tea were administered at 2% (w/v) as the sole source of drinking fluid either 2 wk before and 2 wk during PhIP dosing (100 mg/kg, every other day by oral gavage), or starting 1 wk after the carcinogen and continued until the study was terminated at 16 wk. In the former protocol, each tea produced marginal inhibition of colonic ACF, despite evidence for changes in several hepatic enzymes involved in heterocyclic amine metabolism. Post-initiation, however, the data were as follows (ACF/colon, mean +/- SE): PhIP/water 12.2 +/- 1.5; PhIP/white tea 5.9 +/- 0.9 (** P < 0.01); PhIP/caffeine 5.9 +/- 1.5 (** P < 0.01); PhIP/EGCG 3.5 +/- 0.8 (***P < 0.001); PhIP/green tea 8.9 +/- 1.2 (P = 0.22, not significant). In the latter study, apoptosis was determined using in situ oligo ligation and cleaved caspase-3 assays, whereas cell proliferation was assessed via bromodeoxyuridine (BrdU) incorporation. No consistent changes were seen in apoptosis assays, but BrdU labeling was as follows (percent of cells positive/colonic crypt, mean +/- SE): PhIP/water 10.4 +/- 0.6; PhIP/white tea 8.6 +/- 0.2 (*P < 0.05); PhIP/EGCG 6.0 +/- 0.85 (** P < 0.01); PhIP/caffeine 8.75 +/- 0.45 (*P < 0.05); PhIP/green tea 9.5 +/- 0.4 (P > 0.05, not significant). The data imply that white tea, caffeine, and EGCG may be most effective post-initiation, via the inhibition of cell proliferation in the colon and through the suppression of early lesions.

Gene expression analysis during tumor enhancement by the dietary phytochemical, 3,3'-diindolylmethane, in rainbow trout.

Indole-3-carbinol (I3C) and 3,3'-diindolylmethane (DIM), a primary I3C derivative, are known dietary chemopreventive agents also available as supplements. However, I3C has been found to act as a tumor promoter in rat (multi-organ) and trout (liver) models. I3C and DIM were previously found to be estrogenic in trout liver based on toxicogenomic profiles. In this study, we compare the post-initiation effects of DIM and 17beta-estradiol (E2) on aflatoxin B(1) (AFB(1))-induced hepatocarcinogenesis in trout. Trout were initiated as embryos with AFB(1) and juvenile fish were fed diets containing 0, 120 or 400 p.p.m. DIM or 5 p.p.m. E2 for 18 weeks. Tumor incidence was determined at 13 months and found to be significantly elevated in AFB(1)-initiated trout fed either 400 p.p.m. DIM or 5 p.p.m. E2 compared with control animals. To evaluate the mechanism of tumor enhancement, hepatic gene expression profiles were examined in animals fed promotional diets during the course of tumorigenesis and in hepatocellular carcinomas (HCCs) of initiated animals. We demonstrate that DIM alters gene expression profiles similar to E2 in liver samples during tumorigenesis and in HCC tumors. Further, HCCs from animals on DIM and E2 promotional diets had a transcriptional signature indicating decreased invasive or metastatic potential compared with HCCs from control animals. Overall, these findings are the first to demonstrate tumor promotion by DIM. They confirm the importance of estrogenic signaling in the mechanism of promotion by dietary indoles in trout liver and indicate a possible dual effect that enhances tumor incidence and decreases potential for metastasis.

Natural chlorophyll inhibits aflatoxin B1-induced multi-organ carcinogenesis in the rat.

Chemoprevention by chlorophyll (Chl) was investigated in a rat multi-organ carcinogenesis model. Twenty-one male F344 rats in three gavage groups (N = 7 rats each) received five daily doses of 250 microg/kg [(3)H]-aflatoxin B(1) ([(3)H]-AFB(1)) alone, or with 250 mg/kg chlorophyllin (CHL), or an equimolar amount (300 mg/kg) of Chl. CHL and Chl reduced hepatic DNA adduction by 42% (P = 0.031) and 55% (P = 0.008), respectively, AFB(1)-albumin adducts by 65% (P < 0.001) and 71% (P < 0.001), respectively, and the major AFB-N(7)-guanine urinary adduct by 90% (P = 0.0047) and 92% (P = 0.0029), respectively. To explore mechanisms, fluorescence quenching experiments established formation of a non-covalent complex in vitro between AFB(1) and Chl (K(d) = 1.22 +/- 0.05 microM, stoichiometry = 1Chl:1AFB(1)) as well as CHL (K(d) = 3.05 +/- 0.04 microM; stoichiometry = 1CHL:1AFB(1)). The feces of CHL and Chl co-gavaged rats contained 137% (P = 0.0003) and 412% (P = 0.0048) more AFB(1) equivalents, respectively, than control feces, indicating CHL and Chl inhibited AFB(1) uptake. However, CHL or Chl treatment in vivo did not induce hepatic quinone reductase (NAD(P)H:quinone oxidoreductase) or glutathione S-transferase (GST) above control levels. These results are consistent with a mechanism involving complex-mediated reduction of carcinogen uptake, and do not support a role for phase II enzyme induction in vivo under these conditions. In a second study, 30 rats in three experimental groups were dosed as in study 1, but for 10 days. At 18 weeks, CHL and Chl had reduced the volume percent of liver occupied by GST placental form-positive foci by 74% (P < 0.001) and 77% (P < 0.001), respectively compared with control livers. CHL and Chl reduced the mean number of aberrant crypt foci per colon by 63% (P = 0.0026) and 75% (P = 0.0004), respectively. These results show Chl and CHL provide potent chemoprotection against early biochemical and late pathophysiological biomarkers of AFB(1) carcinogenesis in the rat liver and colon.

The importance of carcinogen dose in chemoprevention studies: quantitative interrelationships between, dibenzo[a,l]pyrene dose, chlorophyllin dose, target organ DNA adduct biomarkers and final tumor outcome.

Chlorophyllin (CHL) is a potent antimutagen in vitro, an effective anti-carcinogen in several animal models, and significantly reduced urinary biomarkers of aflatoxin B(1) (AFB(1)) exposure in a human population. Here we report an expanded analysis of CHL chemoprevention using the potent environmental hydrocarbon dibenzo[a,l]pyrene (DBP). A dose-dose matrix design employed over 12 000 rainbow trout to evaluate the interrelationships among dietary carcinogen dose, anti-carcinogen dose, carcinogen-DNA adduct levels at exposure and eventual tumor outcome in two target organs. Included was an evaluation of the pharmaceutical CHL preparation (Derifil), used previously in a study of individuals chronically exposed to AFB(1). CHL was pre-, co- and post-fed at doses of 0-6000 p.p.m. and co-fed with DBP at doses of 0-371.5 p.p.m. for 4 weeks. This protocol generated a total of 21 dose-dose treatment groups, each evaluated with three or more replicates of 100 animals. The DBP-only treatment produced dose-responsive increases in liver and stomach DBP-DNA adducts, whereas increasing CHL co-treatment doses produced successive inhibition in liver (49-83%) and stomach (47-75%) adduct levels at each DBP dose examined. The remaining 8711 trout were necropsied, 10 months later. DBP treatment alone produced a logit incidence versus log [DBP] dose-response curve in stomach that was linear; CHL co-treatment provided dose-dependent tumor inhibition which ranged from 30 to 68% and was predictable from the adduct response. The Derifil CHL preparation was also found to effectively reduce DNA adduction and final tumor incidence in stomach (as well as liver), with a potency compatible with its total chlorin content. Liver tumor incidence in the DBP-only groups appeared to plateau near 60%. At DBP doses of

Antioxidant and antigenotoxic activities of Angelica keiskei, Oenanthe javanica and Brassica oleracea in the Salmonella mutagenicity assay and in HCT116 human colon cancer cells.

Epidemiological studies indicate that consumption of green-yellow vegetables rich in chlorophyll, vitamin C, vitamin E, and carotenoids reduce the risk of cancer. We sought to examine the antigenotoxic and antioxidant properties of chlorophyll-rich methanol extracts of Angelica keiskei, Oenanthe javanica, and Brassica oleracea (kale). In the Salmonella mutagenicity assay, A. keiskei caused dose-dependent inhibition against three heterocyclic amine mutagens in the presence of S9, O. javanica was antimutagenic only at the highest concentration in the assay (2 mg/plate), and B. oleracea showed no consistent inhibitory activity at non-toxic levels. None of the extracts were effective against three direct-acting mutagens in the absence of S9. Extracts of A. keiskei and, to a lesser extent O. javanica, inhibited two of the major enzymes that play a role in the metabolic activation of heterocyclic amines, based on ethoxyresorufin-O-deethylase and methoxyresorufin-O-demethylase assays in vitro. All three plant extracts were highly effective in assays which measured ferric reducing/antioxidant power, oxygen radical absorbance capacity, and Fe2+/H2O2-mediated DNA nicking. Finally, using the 'comet' assay, all three plant extracts protected against H2O2-induced genotoxic damage in human HCT116 colon cancer cells. These findings provide support for the antigenotoxic and antioxidant properties of chlorophyll-rich extracts of A. keiskei, O. javanica, and B. oleracea, through mechanisms that include inhibition of carcinogen activation and scavenging of reactive oxygen species.

Tumors from rats given 1,2-dimethylhydrazine plus chlorophyllin or indole-3-carbinol contain transcriptional changes in beta-catenin that are independent of beta-catenin mutation status.

Tumors induced in the rat by 1,2-dimethylhydrazine (DMH) contain mutations in beta-catenin, but the spectrum of such mutations can be influenced by phytochemicals such as chlorophyllin (CHL) and indole-3-carbinol (I3C). In the present study, we determined the mutation status of beta-catenin in more than 50 DMH-induced colon tumors and small intestine tumors, and compared this with the concomitant expression of beta-catenin mRNA using quantitative real-time RT-PCR analysis. In total, 19/57 (33%) of the tumors harbored mutations in beta-catenin, and 14/19 (74%) of the genetic changes substituted amino acids adjacent to Ser33, a key site for phosphorylation and beta-catenin degradation. These tumors were found to express a 10-fold range of beta-catenin mRNA levels, independent of the beta-catenin mutation status and phytochemical exposure, i.e. CHL or I3C given post-initiation. However, beta-catenin mRNA levels were strongly correlated with mRNA levels of c-myc, c-jun and cyclin D1, which are targets of beta-catenin/Tcf signaling. Tumors with the highest levels of beta-catenin mRNA often had over-expressed beta-catenin protein, and those with lower beta-catenin mRNA typically had low beta-catenin protein expression, but there were exceptions (high beta-catenin mRNA/low beta-catenin protein, or vice versa). We conclude that DMH-induced mutations stabilize beta-catenin protein in tumors, which increase c-myc, c-jun and cyclin D1, but there also can be over-expression of beta-catenin itself at the mRNA level, contributing to high beta-catenin protein levels. Similar findings have been reported in primary human colon cancers and their liver metastases, compared with matched normal-looking tissue. Thus, further studies are warranted on the mechanisms that upregulate beta-catenin at the transcriptional level in human and rodent colon cancers.

Indole-3-carbinol in the maternal diet provides chemoprotection for the fetus against transplacental carcinogenesis by the polycyclic aromatic hydrocarbon dibenzo[a,l]pyrene.

The fetus and neonate are sensitive targets for chemically induced carcinogenesis. Few studies have examined the risk/benefit of chemoprotective phytochemicals, given in the maternal diet, against transplacental carcinogenesis. In this study, B6129 SF1/J (AHR(b-1/d)) and 129Sv/ImJ (AHR(d/d)) mice were cross-bred. The polycyclic aromatic hydrocarbon, dibenzo[a,l]pyrene (DBP), was administered to pregnant mice (15 mg/kg, gavage) on gestation day 17, and 2000 p.p.m. indole-3-carbinol (I3C), a chemoprotective phytochemical from cruciferous vegetables, was fed to half of the mice from gestation day 9 until weaning. Offspring born to dams fed I3C exhibited markedly fewer mortalities (P < 0.0001). Maternal dietary exposure to I3C also significantly lowered lung tumor multiplicity (P = 0.035) in offspring surviving to 10 months of age. The I3C chemoprotection was independent of either maternal or fetal AHR genotype. The bioavailability of DBP to fetal target tissue was demonstrated by assessing DNA covalent adduction with a (33)P-post-labeling assay. The bioavailability of I3C was determined by dosing a subset of pregnant mice with [(14)C]-I3C. Addition of chemoprotective agents to the maternal diet during pregnancy and nursing may be an effective new approach in reducing the incidence of cancers in children and young adults.

Post-initiation chlorophyllin exposure does not modulate aflatoxin-induced foci in the liver and colon of rats.

Chlorophyllin (CHL) is a promising chemopreventive agent believed to block cancer primarily by inhibiting carcinogen uptake through the formation of molecular complexes with the carcinogens. However, recent studies suggest that CHL may have additional biological effects particularly when given after the period of carcinogen treatment. This study examines the post-initiation effects of CHL towards aflatoxin B1 (AFB1)-induced preneoplastic foci of the liver and colon. The single concentration of CHL tested in this study (0.1% in the drinking water) had no significant effects on AFB1-induced foci of the liver and colons of rats.

In utero exposure of mice to dibenzo[a,l]pyrene produces lymphoma in the offspring: role of the aryl hydrocarbon receptor.

Lymphoma and leukemia are the most common cancers in children and young adults; in utero carcinogen exposure may contribute to the etiology of these cancers. A polycyclic aromatic hydrocarbon (PAH), dibenzo[a,l]pyrene (DBP), was given to pregnant mice (15 mg/kg body weight, gavage) on gestation day 17. Significant mortalities in offspring, beginning at 12 weeks of age, were observed due to an aggressive T-cell lymphoblastic lymphoma. Lymphocytes invaded numerous tissues. All mice surviving 10 months, exposed in utero to DBP, exhibited lung tumors; some mice also had liver tumors. To assess the role of the aryl hydrocarbon receptor (AHR) in DBP transplacental cancer, B6129SF1/J (AHR(b-1/d), responsive) mice were crossed with strain 129S1/SvIm (AHR(d/d), nonresponsive) to determine the effect of maternal and fetal AHR status on carcinogenesis. Offspring born to nonresponsive mothers had greater susceptibility to lymphoma, irrespective of offspring phenotype. However, when the mother was responsive, an AHR-responsive phenotype in offspring increased mortality by 2-fold. In DBP-induced lymphomas, no evidence was found for TP53, beta-catenin, or Ki-ras mutations but lung adenomas of mice surviving to 10 months of age had mutations in Ki-ras codons 12 and 13. Lung adenomas exhibited a 50% decrease and a 35-fold increase in expression of Rb and p19/ARF mRNA, respectively. This is the first demonstration that transplacental exposure to an environmental PAH can induce a highly aggressive lymphoma in mice and raises the possibility that PAH exposures to pregnant women could contribute to similar cancers in children and young adults.