Search

Chondroitin sulfate (CS) is the placental receptor for the VAR2CSA malaria protein, expressed at the surface of infected erythrocytes during Plasmodium falciparum infection. Infected cells adhere to syncytiotrophoblasts or get trapped within the intervillous space by binding to a determinant in a 4-O-sulfated CS chains. However, the exact structure of these glycan sequences remains unclear. VAR2CSA-reactive CS is also expressed by tumor cells, making it an attractive target for cancer diagnosis and therapeutics. The identities of the proteoglycans carrying these modifications in placental and cancer tissues remain poorly characterized. This information is clinically relevant since presentation of the glycan chains may be mediated by novel core proteins or by a limited subset of established proteoglycans. To address this question, VAR2CSA-binding proteoglycans were affinity-purified from the human placenta, tumor tissues and cancer cells and analyzed through a specialized glycoproteomics workflow. We show that VAR2CSA-reactive CS chains associate with a heterogenous group of proteoglycans, including novel core proteins. Additionally, this work demonstrates how affinity purification in combination with glycoproteomics analysis can facilitate the characterization of CSPGs with distinct CS epitopes. A similar workflow can be applied to investigate the interaction of CSPGs with other CS binding lectins as well., (© The Author(s) 2020. Published by Oxford University Press. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.)

CpG endonuclease activity was identified in nuclear extracts obtained from mouse lung tumors. Enzyme activity was determined using a 333 bp polymerase chain reaction product of the estrogen receptor-alpha gene that contained either radiolabeled cytosine or tritium-labeled methyl groups at CpG sites. Activity was measured as the release of radioactivity from the substrate. The product of the nuclease activity was identified by high pressure liquid chromatography (HPLC) as either 5-methyl-2'-deoxycytidine when the CpG sites in the substrate were methylated or 2'-deoxycytidine when the CpG sites were not methylated. The CpG endonuclease activity was dependent on nuclear protein and temperature, had a proclivity for double-stranded over single-stranded DNA and was inhibited by ethylenediaminetetraacetic acid or 2-mercaptoethanol. Strain A/J mouse lung tumors induced by vinyl carbamate had a greater level of CpG endonuclease activity than non-involved lung tissue. Budesonide, a potent chemopreventive agent in mouse lung, not only prevented an increase in CpG endonuclease activity in lung tumors but, when administered to mice with established tumors, also decreased the level of endonuclease activity in the tumors. The effect of budesonide on CpG endonuclease activity in lung tumors was inversely related to its published effect on DNA methylation in mouse lung tumors, i.e. the drug decreased CpG endonuclease activity and increased the methylation of DNA. The increased CpG endonuclease activity in mouse lung tumors and its inhibition by budesonide would suggest this endonuclease as a potential molecular target for chemoprevention.

Budesonide (an anti-inflammatory glucocorticoid), R115777 (a farnesyl transferase inhibitor, Zarnestra, Tipifarnib) or combinations of them were evaluated for prevention of lung tumors and for modulation of DNA methylation in tumors. Lung tumors were induced by vinyl carbamate in female strain A mice. One week later, mice received 60 or 100 mg/kg R115777 by oral gavage and 5 days/week, 0.8 or 1.6 mg/kg of budesonide in their diet, or their combined treatment until killed at 20, 28 and 36 weeks after administering the vinyl carbamate. Other mice were administered the drugs for 2 weeks before killing at Week 20. At Week 20, the rank order for prevention of lung tumors was the combined treatment > budesonide > R115777. At later killings, R115777 was no longer effective, whereas budesonide and the combinations continued to prevent tumors, albeit at a reduced efficacy. DNA hypomethylation in lung tumors was prevented by treatment with R115777, budesonide and the combinations. When administered starting at Week 18 to tumor-bearing mice, the drugs reversed DNA hypomethylation in the tumors. In summary, combined treatment with budesonide and R115777 produced the following results: (i) it was more efficacious in preventing lung tumors than the individual drugs; and (ii) it prevented and reversed DNA hypomethylation in lung tumors. These results support the combined use of budesonide and R115777 in prevention of lung tumors and suggest that reversal of DNA hypomethylation in lung tumors would be useful as a surrogate end-point biomarker for prevention.

Budesonide (an anti-inflammatory glucocorticoid), R115777 (a farnesyl transferase inhibitor, Zarnestra, Tipifarnib) or combinations of them were evaluated for prevention of lung tumors and for modulation of DNA methylation in tumors. Lung tumors were induced by vinyl carbamate in female Strain A mice. One week later, mice received 60 or 100 mg/kg R115777 by oral gavage and 5 days/week, 0.8 or 1.6 mg/kg of budesonide in their diet, or their combined treatment until killed at 20, 28 and 36 weeks after administering the vinyl carbamate. Other mice were administered the drugs for 2 weeks before killing at 20 weeks. At Week 20, the rank order for prevention of lung tumors was the combined treatment>budesonide>R115777. At later killings, R115777 was no longer effective, whereas budesonide and the combinations continued to prevent tumors, albeit at a reduced efficacy. DNA hypomethylation in lung tumors was prevented by treatment with R115777, budesonide and the combinations. When administered starting at Week 18 to tumor-bearing mice, the drugs reversed DNA hypomethylation in the tumors. In summary, combined treatment with budesonide and R115777 produced the following results: (i) it was more efficacious in preventing lung tumors than the individual drugs; and (ii) it prevented and reversed DNA hypomethylation in lung tumors. These results support the combined use of budesonide and R115777 in prevention of lung tumors and suggest that reversal of DNA hypomethylation in lung tumors would be useful as a surrogate endpoint biomarker for prevention.

The ability of celecoxib and alpha-difluoromethylornithine (DFMO) to modulate the DNA hypomethylation and the hypermethylation of the estrogen receptor (ER)-alpha gene in colon tumors was evaluated as potential biomarkers for chemoprevention. Colon tumors were induced in rats by azoxymethane. Celecoxib (500 mg/kg), DFMO (100, 1000 and 3000 mg/kg) or celecoxib + 1000 mg/kg DFMO were administered in the diet for 7 or 28 days prior to death at week 37. Relative to the normal colon mucosa, colon tumors contained global hypomethylated DNA but simultaneous hypermethylation of the promoter plus exon-1 region of the ER-alpha gene. Limited treatment with celecoxib (500 p.p.m. in diet) or DFMO (1000 or 3000 p.p.m. in diet) reversed the DNA hypomethylation. Administering 1000 and 3000 p.p.m. DFMO for 7-days decreased the number of methylated CpG sites in the ER-alpha gene from 5.00 +/- 0.95 to 3.83 +/- 0.75 and 1.75 +/- 0.49 these levels were further reduced to 0.50 +/- 0.26 following administration of 1000 mg/kg for 28 days. Celecoxib administered for 7 and 28 days reduced the number of methylated sites to 4.25 +/- 0.48 and 1.5 +/- 0.50. The combination containing celecoxib and DFMO reduced the number of methylated sites to 0.20 +/- 0.20 at both 7 and 28 days. In parallel with the hypermethylation of the ER-alpha gene, the mRNA expression of the gene was decreased in colon tumors and was increased by celecoxib, DFMO or the combination. Celecoxib and DFMO reversed DNA hypomethylation and the hypermethylation of the ER-alpha gene in colon tumors supporting the hypothesis that modulation of methylation is a biomarker of chemoprevention.

Modulation of gene expression in tumors has the potential of being a surrogate end-point biomarker for chemoprevention. Thus, we determined the modulation by chemopreventive agents of the protein and mRNA expression of genes in rat colon tumors. Male F344 rats were administered three weekly injections of 15 mg/kg azoxymethane. Forty-seven weeks later, they received aspirin (600), calcium chloride (50 000), 2-(carboxyphenyl) retinamide (2-CPR, 315), alpha-difluoromethylornithine (DFMO, 3000), piroxicam (200), quercetin (33 600), 9-cis retinoic acid (9-cis RA, 30), rutin (3000), or sulindac (280) in their diet at the indicated mg/kg concentration for 7 days and were then killed. In colon tumors relative to the mucosa, the protein and mRNA levels of c-myc were increased, while the levels of p16 and p27 were decreased. Calcium chloride, DFMO, piroxicam and sulindac administered for 7 days decreased the mitotic index and reduced the protein and mRNA levels of c-myc in colon tumors. Calcium chloride, DFMO and piroxicam increased the protein and mRNA levels of p16 and along with sulindac increased the protein level of p27, but not its mRNA. The other agents failed to modulate both the mitotic index and the expression of the genes. The ability of the chemopreventive agents to prevent colon tumors was determined. Male F344 rats were administered three weekly injections of 15 mg/kg azoxymethane and 8 weeks later they were administered aspirin, 2-CPR, DFMO, piroxicam, 9-cis RA and rutin in their diet. The rats were killed 26 weeks after they started to receive the chemopreventive agents. The multiplicity of colon tumors was reduced by DFMO and piroxicam, increased by rutin and not affected by the other agents. Hence, agents that prevented colon cancer decreased the mitotic index and altered the expression of c-myc, p16 and p27 suggesting that modulation in the expression of these genes are potential biomarkers for chemopreventive activity.

Chemopreventive drugs have the potential to decrease the morbidity and mortality of lung cancer. The development of these drugs could be expedited by the application of surrogate end-point biomarkers that demonstrate chemopreventive efficacy. In this study, the ability of budesonide to prevent lung tumors in mice was characterized further and its effects on biomarkers were determined. Lung tumors were induced in female strain A mice by vinyl carbamate (16 mg/kg) administered once weekly for 2 consecutive weeks. Four weeks later the mice started to receive 0.6, 1.2 or 2.4 mg/kg budesonide continually in the diet until killed at week 20. Budesonide caused a dose-dependent decrease in the multiplicity of lung tumors of 25, 58 and 82%, respectively. Budesonide (2.4 mg/kg diet) administered starting at weeks 4, 10 or 16, decreased tumor multiplicity by 82, 66 and 30% at week 20. Administering 2.4 mg/kg budesonide at weeks 4-20 or 20-35 and killing the mice at week 35 did not significantly decrease the yield of tumors, although both treatment regimens did decrease the size of the tumors and the progression of adenomas to carcinomas. Thus, budesonide delayed the appearance of lung tumors and decreased their growth and progression to carcinomas. To determine the effect of limited exposure to budesonide on biomarkers, it was administered for only 7 days prior to death at week 35. Budesonide decreased the proliferating cell nuclear antigen labeling in lung adenomas, carcinomas, parenchyma and bronchial airways by 87.6, 59.0, 41.1 and 25.4%, respectively. Budesonide treatment also increased the protein level of the p21 and p27 genes and increased the mRNA level of p21. Thus, short-term treatment with budesonide modulated biological and molecular end-points in lung tumors that might be developed further as biomarkers for its clinical chemopreventive efficacy in the lung.

Chloroform, dichloroacetic acid (DCA) and trichloroacetic acid (TCA) are mouse liver carcinogens that are chlorine disinfection by-products found in drinking water. The effect of chloroform on DCA and TCA-induced hypomethylation and expression of the c-myc gene and on their promotion of liver and kidney tumors was determined. B6C3F1 mice were administered 0, 400, 800 and 1600 mg/l chloroform in drinking water and 500 mg/kg DCA or TCA-administered daily by gavage. DCA, TCA and to a lesser extent chloroform decreased the methylation and increased the mRNA expression of the c-myc gene. Co-administering chloroform prevented only DCA and not TCA-induced hypomethylation and increased mRNA expression of the gene. The effect of chloroform on tumor promotion by DCA and TCA was determined in female and male B6C3F1 mice initiated on day 15 of age with N-methyl-N-nitrosourea. Starting at 5 weeks of age, the mice received in their drinking water DCA (3.2 g/l) or TCA (4.0 g/l) with 0, 800 or 1600 mg/l chloroform until they were killed at 36 weeks. Liver tumors promoted by DCA and TCA were predominantly basophilic except for DCA-treated female mice that were eosinophilic. Only DCA promoted foci of altered hepatocytes and they were eosinophilic in both sexes. Chloroform prevented DCA, but not TCA promotion of liver foci and tumors. In male mice, TCA promoted kidney tumors while DCA promoted kidney tumors only when co-administered with chloroform. Hence, chloroform prevented the hypomethylation and increased mRNA expression of the c-myc gene and the promotion of liver tumors by DCA, while enhancing DCA-promotion of kidney tumors. Thus, the concurrent exposure to two carcinogens, chloroform and DCA resulted in less than additive activity in one organ and synergism in another organ.

The dose-response relationship in male F344 rats was determined for the ability of aspirin administered in the diet to prevent azoxymethane (AOM)-induced colon cancer and aberrant crypt foci (ACF) and to reduce prostaglandin E2 (PGE2) levels. Starting at either 7 or 22 weeks of age, the rats received aspirin. All rats received two doses of AOM (15 mg/kg each on days 7 and 14) and were killed on day 36. The lowest concentrations of aspirin to prevent ACF or reduce PGE2 levels were 600 and 400 mg/kg, respectively. To evaluate the prevention of tumors, rats received either 0 or 400 mg/kg aspirin for a total of 39 weeks with AOM (30 mg/kg) administered 7 days after the start of treatment. Aspirin had no effect on the yield of colon tumors. In a second experiment, rats started to receive 0, 200, 600 or 1800 mg/kg aspirin or 1000 mg/kg alpha-difluoromethylornithine (DFMO) +/- aspirin. Eight and 15 days later, all the rats received 15 mg/kg AOM. Eleven weeks later, animals that were receiving the control diet started to receive 0, 200, 600 or 1800 mg/kg aspirin; 1000 or 3000 mg/kg DFMO; or 1000 mg/kg DFMO + 200 or 600 mg/kg aspirin. The animals were killed 32 weeks later. DFMO effectively reduced the yield of colon tumors when administered starting either before or after AOM while aspirin was much weaker. The combination of aspirin + DFMO administered after AOM was synergistic. Both aspirin and DFMO decreased the Mitotic Index, while apoptosis was increased only by DFMO. Our results demonstrated that aspirin and DFMO could prevent colon cancer when administered after AOM. Furthermore, aspirin reduced ACF, PGE2 levels and mitosis at concentrations that did not prevent cancer. In contrast, the ability to enhance apoptosis did correlate with the prevention of cancer.

Evidence is accumulating which indicates that cyclooxygenase-2 (COX-2) is involved in the pathogenesis of colorectal cancer. We evaluated the expression of COX-2 in replication error-positive (RER) colon cancers, colon cancers metastatic to liver and azoxymethane (AOM)-induced rat colonic tumors. Immunohistochemistry showed that COX-2 was low to undetectable in normal human mucosa, but abundant in the RER adenocarcinomas we examined. COX-2 immunoreactivity in metastatic colon cancers was less abundant, but clearly detectable. In the colon of AOM-treated rats, COX-2 protein was not detectable in normal mucosa, but present in most of the epithelial cells comprising the tumors. The TGF-beta1 staining pattern in these human and rat tumors was similar to that observed for COX-2. The role of TGF-beta in RER adenocarcinomas is complex because of the increased mutation rate of TGF-beta type II receptors. Northern analysis showed abundant TGF-beta1 mRNA in AOM-induced tumors, but not in paired mucosa. TGF-beta1 induced the expression of COX-2 mRNA and protein in intestinal epithelial cells (IEC-6). Chronic TGF-beta1 treatment caused a TGF-beta-dependent overexpression of COX-2 in rat intestinal epithelial cells (RIE-1). TGF-beta1 may regulate COX-2 expression during the colonic adenoma to carcinoma sequence.

We have previously reported that the retinoids, 4-(hydroxyphenyl)retinamide (4-HPR) and 9-cis-retinoic acid (RA) prevented azoxymethane (AOM)-induced colon tumors and along with 2-(carboxyphenyl)retinamide (2-CPR) prevented aberrant crypt foci (ACF). In this study, we evaluated the effect of 2-CPR on AOM-induced colon tumors and the effect of the three retinoids on apoptosis and cell proliferation. Male F344 rats were administrated 15 mg/kg AOM at weeks 7 and 8 of age. 2-CPR (315 mg/kg) was administered in the diet starting either 1 week before or at week 12 after the first dose of AOM. The rats continued to receive the 2-CPR until killed at week 46. Unlike the demonstrated prevention of colon cancer by the other two retinoids, both dosing schedules of 2-CPR resulted in an approximate doubling of the yield of colon tumors. In adenomas, 2-CPR, 4-HPR and 9-cis-RA were equally effective in reducing mitotic activity, while only 4-HPR and 9-cis-RA but not 2-CPR enhanced apoptosis. When administered for only the 6 days prior to killing 4-HPR but not 2-CPR decreased the Mitotic Index and increased the Apoptotic Index in adenomas. In non-involved crypts, chronic exposure to 4-HPR and 9-cis-RA in contrast to 2-CPR reduced the Mitotic Index and enhanced the Apoptotic Index. In concurrence with our previous study, both 2-CPR and 4-HPR were very potent in preventing ACF when administered in the diet starting 1 week before the first dose of AOM and continuing for the 5 weeks of the study. Hence, unlike the other two retinoids, 2-CPR, although very potent in preventing ACF, enhanced rather than prevented AOM-induced colon cancer. Furthermore, our results suggest that the effect of 2-CPR on tumor yield is different from 4-HPR and 9-cis-RA because, unlike them, it does not enhance apoptosis.

The physical interaction between beta-catenin and the adenomatous polyposis coli (APC) gene, and the ability of APC to regulate cytoplasmic levels of beta-catenin suggest a role for beta-catenin in colorectal carcinogenesis. In this study, we found that beta-catenin immunoreactivity was detected exclusively in the cell membrane and cytoplasm of morphologically normal intestinal epithelial cells with predominant distribution in the differentiated nonproliferative cell population. In contrast, beta-catenin was localized predominantly in the nucleus of adenomas from Min/+ mice and transgenic mice expressing a mutant truncated form of the APC gene (Apc(delta716) mice). Beta-catenin was expressed predominantly at the cell membrane and cytoplasm of the nontransformed rat intestinal epithelial (RIE-1) cells in culture, whereas predominantly nuclear localization of beta-catenin was observed in the human colon cancer cell line SW480. In the azoxymethane (AOM) treated rats, overexpression and nuclear localization of beta-catenin was observed in all adenomas. Previous studies have indicated the incidence of APC mutations amongst AOM-induced tumors to be 15% or less. These results demonstrate that nuclear localization of beta-catenin is a common event in colorectal tumorigenesis.

Retinoids are proposed chemopreventive agents that inhibit cell proliferation and induce differentiation. Their ability to prevent azoxymethane (AOM)-induced aberrant crypt foci (ACF) and tumors and to modulate cell proliferation was investigated in the colon of male F344 rats. Thirteen retinoids were evaluated for prevention of ACF and two of them, 9-cis-retinoic acid (RA) and 4-(hydroxyphenyl)retinamide (4-HPR), were also evaluated for prevention of colon cancer. The retinoids were administered continuously in the diet starting 1 week prior to the first of two weekly 15 mg/kg i.p. injections of AOM and for a total of either 5 or 36 weeks in order to evaluate their effect on colonic ACF and tumors. At a concentration of 1 mmol/kg diet, 2-(carboxyphenyl)retinamide caused the greatest reduction (57.7%) in the yield of ACF. 9-cis-RA was toxic at 1 mmol/kg so that it was evaluated at 0.1 mmol/kg, resulting in a 41.6% reduction in ACF. The ability of the retinoids to reduce the proliferating cell nuclear antigen (PCNA) labeling index in ACF and in non-involved crypts correlated with their ability to prevent ACF. Both 9-cis-RA (0.1 and 0.2 mmol/kg diet) and 4-HPR (1 and 2 mmol/kg diet) were highly effective in decreasing the yield of AOM-induced colon tumors. In summary, retinoids were demonstrated to reduce cell proliferation and to prevent ACF and tumors in the colon, suggesting promise as preventive agents for colon cancer.

Curcumin and quercetin were evaluated in rats for their ability to modulate the carcinogenic activity of azoxymethane (AOM) in the colon and 7,12-dimethylbenz[a]anthracene (DMBA) in the mammary gland. In the AOM-induced colon cancer model, male Fischer 344 rats at 8 weeks of age started to receive either curcumin (8 and 16 g/kg) or quercetin (16.8 and 33.6 g/kg) in the diet and 1 week later, were administered AOM (30 mg/kg body wt.) by subcutaneous injection. The animals continued to receive the two agents in the diet until sacrificed 45 weeks later. Curcumin mediated a dose-dependent inhibition of the incidence and multiplicity of adenomas from 47% and 0.58 +/- 0.12 adenomas/rat in the AOM-treated control group to 19% and 0.22 +/- 0.08 and 0.06% and 0.08 +/- 0.06 adenomas/rat for the low and high dose groups, respectively. A low yield of adenocarcinomas (0.06 +/- 0.04 adenocarcinomas/rat) was induced by AOM which was not significantly altered by curcumin. Treatment with quercetin caused a dose-dependent increase in the yield of AOM-induced tumors in the colon from 0.06 +/- 0.04 adenocarcinoma/rat to 0.64 +/- 0.12 and 1.14 +/- 0.17 for the low and high dose groups, respectively. In the DMBA-induced mammary cancer model, curcumin or quercetin was administered at either 10 or 20 g/kg diet, beginning 7 days prior to DMBA and continually throughout the remainder of the experiment. Neither curcumin nor quercetin significantly altered the incidence of animals with tumors or the tumor multiplicity, while the high concentration of both agents significantly increased tumor latency. These results demonstrate different responses to these agents in the two models. While curcumin was highly effective as a chemopreventive agent in the colon model, it was only weakly effective in the mammary model. In contrast, quercetin which was also only weakly effective in the mammary model, caused a dose-dependent enhancement of tumors induced by AOM in the colon model.

Piroxicam has been shown to prevent azoxymethane (AOM)-induced aberrant crypt foci and colon cancer in rats. In this communication we evaluate whether piroxicam can also cause regression of precancerous lesions identified as aberrant crypt foci, thus preventing the occurrence of cancer. Male Fischer-344 rats were administered 0.125 g/kg piroxicam in their diet starting either 1 week prior to or 12 weeks after a single subcutaneous injection of AOM (30 mg/kg body wt). The yield of aberrant crypt foci and of colon adenomas and adenocarcinomas was determined at 5, 12, 27 and 37 weeks after administering the AOM respectively. When piroxicam was administered starting 1 week prior to AOM the yield of aberrant crypt foci at the three initial time points was reduced. When the administration of piroxicam was delayed until 12 weeks after AOM the yield of aberrant crypt foci was reduced from 53.8 +/- 8.1 foci/colon at 12 weeks to 11.1 +/- 2.0 at 27 weeks. At 37 weeks after administering AOM the yield of colon tumors was 0.59 +/- 0.11 tumors/animal, while in rats administered piroxicam beginning either 1 week prior to or 12 weeks after AOM the yield was similarly reduced to 0.14 +/- 0.07 and 0.17 +/- 0.07 tumors/animal respectively. Thus piroxicam was demonstrated not only to prevent, but also to cause regression of aberrant crypt foci, both of which were associated with the prevention of colon tumors.

The purpose of this study was to evaluate the effects of the loss of a p53 allele and phenethyl isothiocyanate (PEITC) pre-treatment on the tumorigenicity of 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK) and K-ras mutation frequency in a hybrid mouse model. Male TSG-p53 'knock-out' mice were bred with A/J female mice to produce (A/J x TSG-p53) F1 mice either homozygous (p53+/+) or heterozygous (p53+/-) for p53 alleles. These mice, together with female A/J mice, were treated at 6-8 weeks of age with NNK or dosed with PEITC prior to administration of NNK. The A/J mice treated with NNK had a 100% incidence of lung tumors, with 9.7 +/- 3.4 tumors/mouse. A/J mice pre-treated with PEITC prior to NNK administration had 3.5 +/- 2.1 lung tumors/animal, although the incidence remained at 100%. In (A/J x TSG-p53) F1 mice with either the p53(+/-) or p53(+/+) genotype PEITC pre-treatment significantly decreased tumor incidence (100 to 40 and 36%, respectively) and multiplicity (2.0 +/- 0.5 to 0.5 +/- 0.4 and 2.1 +/- 0.5 to 0.5 +/- 0.4, respectively), indicating that PEITC is an effective chemopreventive agent in both A/J mice and (A/J x TSG-p53) F1 mice. Analysis of lung tumor DNA from A/J mice treated with NNK or NNK/PEITC indicated that 15 of 17 (88%) and 20 of 23 (87%) of the tumors, respectively, contained G-->A transitions at the second base of codon 12 in the K-ras gene. Similarly, in lung tumors from (A/J x TSG-p53) F1 mice treated with NNK or NNK/PEITC 29 of 30 (96%) and 9 of 10 (90%), respectively contained G-->A transitions at the second base of codon 12 of the K-ras gene. No mutations of the p53 gene were found in any of the tumors analyzed, suggesting minimal involvement of this gene in the development of lung adenomas. These data indicate that absence of a p53 allele in (A/J x TSG-p53) F1 mice does not alter the incidence or multiplicity of NNK-induced lung tumors and that PEITC inhibition of NNK tumorigenesis does not affect the frequency or spectrum of K-ras gene mutations found consistently with NNK carcinogenesis.

The frequency and mutation spectra of proto-oncogene activation in hepatocellular neoplasms induced by tetrachloroethylene, trichloroethylene and dichloroacetic acid were examined to help define the molecular basis for their carcinogenicity. H-ras codon 61 activation was not significantly different among dichloroacetic acid- and trichloroethylene-induced and combined historical and concurrent control hepatocellular tumors (62%, 51% and 69% respectively). The mutation spectra of H-ras codon 61 mutations showed a significant decrease in AAA and increase in CTA mutations for dichloroacetic acid- and trichloroethylene-induced tumors when compared to combined controls. The H-ras codon 61 mutation frequency for tetrachloroethylene-induced tumors was significantly lower (24%) than that of combined controls and also that of the two other chemicals. Mutations at codons 13 and 117 plus a second exon insert contributed 4% to the total H-ras frequencies for trichloroethylene and tetrachloroethylene. There was also a higher incidence of K-ras activation (13%) in tetrachloroethylene-induced tumors than in the other chemically induced or control tumors. Four liver tumors were found to contain insertions of additional bases within the second exon of K- or H-ras. These findings suggest that exposure to dichloroacetic acid, trichloroethylene and tetrachloroethylene provides a selective growth advantage to spontaneously occurring mutations in codon 61 of H-ras and, at the same time, is responsible for a small number of unique molecular lesions suggestive of either a random genotoxic mode of action or a non-specific result of secondary DNA damage. However, the absence of ras activation in many of the liver neoplasms suggests that alternative mechanisms are also important in B6C3F1 mouse hepatocarcinogenesis.

Foci of aberrant and/or hexosaminidase-negative crypts in rat colon are putative precancerous lesions that have been proposed as biomarkers for short-term bioassays for chemical carcinogens and chemopreventive agents. The ability of a substance to reduce the yield of azoxymethane (AOM)-induced foci in the colon of male Fischer 344 rats, was evaluated as a screening assay for chemopreventive agents. Twenty-eight test agents were administered continuously in the diet from the start of the experiments until the animals were killed 35 days later. AOM was s.c. administered either as 15 mg/kg body wt on days 7 and 14 or as 30 mg/kg body wt on day 7 of the experiment. Foci of aberrant crypts were evaluated in whole mounts of methylene blue-stained colons. AOM induced twice as many foci when administered between 8.40 and 11.00 a.m. than between 2.45 and 5.55 p.m. Calcium salts of carbonate, chloride and glucarate decreased the yield of AOM-induced foci while the acidic salts of lactate and phosphate did not inhibit the formation of foci. Dimethyl-fumarate, fumaric acid, genistein, piroxicam, simethicone, sodium suramin and sulindac reduced the yield of AOM-induced foci of aberrant crypts, with genistein being the most potent. Only piroxicam of this group has previously been shown to inhibit colon cancer, while the rest have yet to be evaluated. Ibuprofen did not inhibit the formation of foci, although it has been reported to inhibit AOM-induced colon cancer in rats. Piroxicam and sulindac appeared to reduce preferentially hexosaminidase-negative foci of aberrant crypts, compared with those of apparently normal morphology. The AOM-induced foci of aberrant crypts assay appears suitable for screening chemicals for chemopreventive action.

The ability of diethylnitrosamine (DENA) to initiate and phenobarbital to promote altered-hepatocyte foci and hepatocellular carcinomas in C3H and C3B6F1 (C3H x C57BL/6) mice was compared to the extent of cell proliferation. Fifteen day old female mice received DENA (16 mg/kg, i.p.) or the vehicle control followed at weaning with 500 p.p.m. sodium phenobarbital in their drinking water until killed at 161 days of age. Six days prior to death each mouse had implanted an osmotic minipump containing 30 mg/ml bromodeoxyuridine in order to measure cell proliferation as the labeling index of bromodeoxyuridine incorporation into newly replicated DNA. C3H mice were more susceptible to DENA than C3B6F1 mice, 54.1 versus 0.57 adenomas/mouse respectively, and phenobarbital increased the yield of altered-hepatocyte foci in C3H mice. Hepatocellular adenomas had a greater bromodeoxyuridine labeling index than altered-hepatocyte foci, which was greater than non-involved/normal hepatocytes. In DENA-initiated C3H and C3B6F1 mice, phenobarbital increased the labeling index in eosinophilic foci, while decreasing the labeling index in normal/non-involved hepatocytes with/without DENA initiation. However, the level of cell proliferation in normal/non-involved hepatocytes, foci and adenomas was lower in C3H mice than in C3B6F1 mice. Thus, the level of cell proliferation did not correlate to the higher yield of foci and tumors in C3H mice. The results are consistent with the strain sensitivity to DENA-initiation and to phenobarbital-promotion correlating to the ratio of the labeling index in the foci to the labeling index in non-involved hepatocytes. An increase in this labeling index ratio for cell proliferation indicates that the foci are selectively stimulated to proliferate resulting in a greater expansion of the population of precancerous cells in the liver.

The effects of phenobarbital (PB) on hepatocellular carcinogenesis in three strains of nitrosamine-initiated infant male mice were evaluated. Fifteen-day-old C57Bl/6NCrlBR (C57Bl), C3H/HeNCr1BR (C3H) and B6C3F1 mice were treated with a single i.p. injection of either diethylnitrosamine (DENA) (5 micrograms/body wt), dimethylnitrosamine (DMNA) (5 micrograms/body wt) or saline. One-half of the treated mice received PB via the drinking water (500 mg/l) for 24 weeks. The remaining treated mice were given deionized drinking water. Mice were killed at 28 weeks of age and hepatic lesions were evaluated. Only animals that received DENA or DMNA exhibited tumors. C3H mice treated with DENA + PB demonstrated a significant increase in hepatic adenoma number compared to C3H mice exposed to DENA only. Conversely, B6C3F1 males treated with DENA + PB exhibited a significant decrease in the number of hepatic adenomas compared to B6C3F1 males treated with DENA alone. No change was noted in adenoma size in B6C3F1 mice treated with DENA + PB from those receiving DENA only. Chronic PB exposure of C57Bl males previously treated with DENA had no effect on hepatic adenoma number or size. C3H mice treated with DMNA + PB displayed an increase in both adenoma size and adenoma number compared to C3H mice receiving DMNA only. Similarly, in B6C3F1 mice, PB treatment increased both the adenoma incidence and adenoma number in DMNA initiated mice. PB had no effect on hepatic adenoma incidence or number in DMNA-treated C57Bl mice. These data suggest that the ability of PB to promote hepatic tumorigenesis in the 15-day-old initiated mouse is dependent on both the strain of the mouse and the initiating chemical carcinogen.

The effect of subsequent administration of chloroform or phenobarbital on the incidence of ethylnitrosourea (ENU) initiated liver and lung tumors was investigated. Fifteen day old Swiss mice were administered ENU, and at weaning they started to receive either 1800 p.p.m. chloroform or 500 p.p.m. sodium phenobarbital in their drinking water. The mice continued to receive either chloroform or phenobarbital until 51 weeks of age. They were sacrificed at 52 weeks of age. ENU at 5 and 20 mg/kg, caused a dose-dependent increase in liver and lung tumors. The male mice were more sensitive to the induction of liver tumors, while no sex preference was observed for the induction of lung tumors. In male mice chloroform inhibited, while in female and male mice phenobarbital promoted spontaneous and ENU-induced liver tumors. Subsequent treatment with either chloroform or phenobarbital did not affect the incidence of ENU-induced lung tumors. In conclusion, when administered in the drinking water, chloroform inhibited while phenobarbital promoted hepatocarcinogenesis in mice.

The induction of gamma-glutamyltranspeptidase (GGT)-foci by single and by split doses of diethylnitrosamine (DENA) was evaluated in the livers of juvenile and young adult male, Sprague-Dawley rats. A single dose of DENA was administered at either 32, 41, 46, 52 or 56 days of age and followed by a promoting regimen of sodium phenobarbital in the drinking water. In 32 days old rats, DENA resulted in a higher incidence of GGT-foci/cm3 than was observed when it was administered to 41 or 46 day old rats. The same dose of DENA was inactive in 52 or 60 day old rats. In rats initiated with DENA on day 32 of age, starting the phenobarbital promotion at 41 days of age resulted in a higher incidence of GGT-foci than when the phenobarbital treatment was begun at 60 days of age. When the dose of DENA was split into five daily doses of DENA starting on day 32 of age, the incidence of GGT-foci/cm3 was equivalent to the single dose given on day 32 of age. However, when the five daily doses were started on 48 days of age, the incidence of GGT-foci/cm3 was less than the five doses of DENA started on day 32 of age. Hence, juvenile rats were more sensitive to both single and split doses of DENA than adult rats. The dose-response relationship was also determined in juvenile rats for a single and for up to 64 daily doses of 3 mg/kg DENA started on day 32 of age. The dose-response relationship for a single dose of 3-96 mg/kg DENA was equivalent to the relationship of one to 32 daily 3 mg/kg doses. Thus, low daily doses of DENA were additive when started in juvenile rats. Neither a threshold nor a plateau was obtained for the dose-response relationship of DENA initiation of GGT-foci.

The indication of ornithine decarboxylase (ODC) by barbiturates and the ability of barbiturates to enhance neoplastic progression of chemically initiated cancer was examined in rat liver. All seven barbiturates induced ODC with barbital (7.7 fold increase) and phenobarbital (5.7 fold increase) demonstrating the most potent activity. Maximum induction of ODC by phenobarbital was obtained in 18 h. Barbital (500-5000 p.p.m.) and phenobarbital (500 p.p.m.) administered in the drinking water enhanced the appearance of diethylnitrosamine (DENA)-initiated gamma- glutamyltranspeptidase (GGTase)-positive foci. Amobarbital, hexabarbital and pentabarbital did not enhance the appearance of GGTase-positive foci. In the absence of previous initiation by DENA, the enhancing regimen of the barbiturates did not cause the appearance of GGTase-positive foci. Barbiturates induced ODC activity in rat liver and enhanced the incidence of DENA initiated GGTase-positive foci.