O6-methylguanine accumulates in DNA of mammary glands after administration of N-methyl-N-nitrosourea to rats.

N-Methyl-N-nitrosourea (MNU) induces mammary carcinoma in female rats when given intravenously. After a single intravenous dose of N-methyl-N-nitrosourea (5 mg/100 g body wt.), we were unable to detect a shift of rat mammary gland DNA on an alkaline sucrose gradient. However, the alkylated products in DNA, 7-methylguanine and O6-methylguanine, were determined at various times following treatment with N-methyl-N-nitrosourea. O6-Methylguanine was removed from the DNA at a slower rate than 7-methylguanine and increased in the DNA with a second injection of N-methyl-N-nitrosourea. 3-Methyladenine was not detected in DNA from the mammary gland of the rat. These data support previous work with brain and bladder that suggest the persistence of O6-methylguanine in DNA might be involved in the induction of cancer by N-methyl-N-nitrosourea.     

Hypomethylation of DNA in Raji cells after treatment with N-methyl-N-nitrosourea

Human Raji lymphoblast-like cells were propagated in the presenceof various concentrations of N-methyl-N-nitrosourea (MNU) andthe degree of enzymatic methylation of newly synthesized DNAwas analysed by two independent methods. The overall extentof enzymatic DNA methylation was measured on the basis of [¹⁴C]deoxycytidinederived radioactivity incorporated into DNA 5-methylcytosineand cytosine residues. Enzymatic methylation of internal cytosinesat 5'- CCGG-3' sequences of Raji DNA was analysed by use ofthe bacterial restriction enzyme Hpall and its isoschizomerMspl. The data obtained by both methods indicate that the treatmentwith MNU causes a lower level of enzymatic methylation of newlysynthesized DNA. This lower extent of DNA methylation persistsin the absence of the carcinogen in the cell cycles followingthe treatment.     

1 Alpha-hydroxyvitamin D3 suppresses colonic tumorigenesis induced by repetitive intrarectal injection of N-methyl-N-nitrosourea in rats

The effect of 1 alpha-hydroxyvitamin D3 (1 alpha (OH)D3) on colonic tumorigenesis induced by chronic treatment with N-methyl-N-nitrosourea (MNU) was studied in rats. Seventy-four female F344 rats received an intrarectal injection of 1 mg of MNU once a week for 40 weeks. Two-thirds of rats were given concomitant administration of 0.2 ml of medium chain triglyceride (MCT) or MCT containing 0.04 microgram of 1 alpha (OH)D3 through an intragastric route thrice weekly. Numbers of rats bearing colonic tumor were 21 in MNU alone (n = 24), 17 in MNU + MCT (n = 25) and 12 in MNU + 1 alpha (OH)D3 group (n = 25) (uncorrected chi 2 = 8.72). The result indicated that colonic tumorigenesis induced by the chronic treatment with MNU was suppressed by oral supplementation of 1 alpha (OH)D3 and the inhibitory effect of 1 alpha (OH)D3 was partly due to the effect of MCT.     

Organ specific modifying potential of ethinyl estradiol on carcinogenesis initiated with different carcinogens

Estrogen has been shown to exert a modifying potential on carcinogenesisin various organs, and in particular the hormone-dependent tissues.The present experiments were carried out to detennine the effectsof post-initiation phase administration of etbinyl estradiol(EE) on tumor development in the liver, kidney, lung, thyroid,bladder and esophagus of male E344 rats. Animals were initiatedby 2 weeks treatment with 0.05% N-bis(2-hydroxypropyl)nitrosamine(DHPN), 0.1% N-ethyl-N-hydroxyethylnitrosamine (EHEN), 0.03%N-nitrosopiperidine (NPD), 0.02% 2-acetyl-aminofluorene (2-AAF)or 0.05% N-butyl-N-(4-hydroxy-butyl)nitrosamine (BBN) in theirdiet or drinking water, and starting 1 week after initiation,they were given diet with or without a 0.001% EE supplementfor 49 weeks, at which point the experiment was terminated.EE significantly increased the development of tumors of theliver (DHPN-, EHEN-, 2-AAF-and NPD-treated groups) and kidneys(EHEN-treated group) but inhibited their development in thelungs (DHPN treated group) and urinary bladder (BBN-treatedgroup). In the liver, EE also increased the development of glutathioneS-transferase P type-positive lesions to various extents dependingon the initiator used. EE administration was associated witha decreased incidence of esophageal hyperplasia in the EHEN-initlatedgroup but an opposite increase in the NPD initiated animals.No effect of EE was evident regarding thyroid lesions.     

Carcinogenic potential of some pesticides in a medium-term multi-organ bioassay in rats

The carcinogenic potential of 5 pesticides was analyzed using a medium-term multi-organ bioassay for carcinogenicity. Male F344 rats were initially treated with 3 known carcinogens (diethylnitrosamine, N-methyl-N-nitrosourea and N-bis(2-hydroxypropyl)nitrosamine) during a period of 4 weeks to induce neoplastic changes in a variety of organs, and then given one of 5 pesticides in the diet for a further 16 weeks. Neoplastic and pre-neoplastic lesions were found in the thyroid, kidney and urinary bladder with propineb, in the forestomach, kidney and thyroid with captan and folpet. The number of glutathione S-transferase placental-form-positive liver-cell foci was significantly increased in the captan- and phosmet-treated groups. Based on these findings, captan and propineb can be considered as carcinogens and carcinogenicity is suspected for folpet and phosmet. These results are in concordance with reported long-term carcinogenicity for captan, folpet and propineb. Daminozide was considered not to be carcinogenic. Thus, the present assay of 20 weeks' duration is useful for the prediction of potential carcinogens.     

Enhancing effects of various gastric carcinogens on development of pepsinogen-altered pyloric glands in rats

To assess the possibility of establishing an in vivo, medium-term bioassay system for gastric carcinogens and promoters, a total of 220 male WKY rats were divided into two groups. Group 1 animals were treated first with a single dose of N-methyl-N'-nitro-N-nitrosoguanidine (MNNG) (160 mg/kg body wt) and starting 2 weeks later administrated one of five gastric carcinogens, a gastric promoter, one of five non-gastric carcinogens or no treatment, as a control, for 14 weeks. Saturated sodium chloride solution (1 ml) treatments were given by gastric intubation at weeks 4, 6, 8 and 10. Group 2 rats received 1 ml of DMSO instead of MNNG and were then treated in the same way as group 1. Analysis of pyloric mucosa sections for pepsinogen altered pyloric glands (PAPG) detected immunohistochemically after the animals were killed at week 16 revealed increased lesion numbers in group 1, with all gastric carcinogens and promoters examined. However, none of the five non-gastric carcinogens exerted any significant modification of PAPG development. The results strongly suggest that the experimental protocol consisting of the following four components: (i) adoption of PAPG as the end-point marker lesion; (ii) single dose of MNNG as initiator; (iii) test chemical administration for 14 weeks; and (iv) administration of saturated sodium chloride solution during the test chemical exposure, could be used effectively for the detection of gastric carcinogens as well as promoters of gastric carcinogenesis in a relatively short time period.     

Effect of pretreatment of rats with N-methyl-N-nitrosourea on the repair of O6-methylguanine in liver DNA

The capacity of rat liver to repair O⁶-methylguanine was determinedby measuring the amounts of this base present in DNA after administrationof N-[¹⁴C]methyl-N-nitrosourea (MNU). Chronic pretreatment ofanimals with unlabelled MNU either once weekly for 5 weeks ordaily on weekdays for 14 days had either no marked effect orinhibited (or overloaded) repair activity, respectively. Pretreatmentof rats with single doses of MNU initially decreased O⁶-methylguaninerepair but this effect was lost as the pretreatment intervalincreased and by 40 h, activity had returned to control values.Administration of methylmethanesulphonate for 14 days had noeffect on the removal of O^6-methylguanine produced in liverDNA by [¹⁴C]MNU. None of the pretreatment schedules inhibitedthe removal of 3-methyladenine from liver DNA. These effectsare contrasted with those produced by pretreatment of rats withdimethylnitro-samine (DMN) or 1, 2-dimethylhydrazine which enhancedthe capacity of the liver to remove O⁶-methylguanine producedby single dose of [¹⁴C]DMN.     

Strong promoting activity by uracil on urinary bladder carcinogenesis and a possible inhibitory effect on thyroid tumorigenesis in rats initiated with N-methyl-N-nitrosourea

Uracil has been shown to cause a strong proliferative responsein the urinary bladder epithelium of rats and mice through calculusformation and, consequently, acts as a strong promoter in bladdercarcinogenesis. In this study, we examined the effect of uracilon two-stage carcinogenesis in various organs using N-methyl-N-nitrosourea(MNU) as the initiator. F344 rats were injected i.p. with MNUtwice weekly for 4 weeks and then given diet containing 3.0%uracil for 20 weeks. Uracil induced urinary bladder carcinomasin all rats pretreated with MNU, and it decreased the combinedincidence of adenomas and carcinomas in the thyroid. Althoughnot significant, uracil decreased the incidence of adenomasin the lung and increased that of lymphomas of the thymus. Apossible influence of a significantly decreased body weightgain caused by uracil treatment on the reduced tumor incidencein the thyroid and lung is discussed. The present data demonstratethe strong promoting activity of uracil for urinary bladdercarcinogenesis, and suggest a possible inhibitory effect onthyroid carcinogenesis.     

Psychological distress after initial treatment of breast cancer. Assessment of potential risk factors.

BACKGROUND. Patient and disease characteristics are often mentioned by clinicians as possible risk factors for psychological distress among women with breast cancer. However, either these factors have not been evaluated or when they were evaluated, results were inconclusive. METHODS. Potential risk factors for psychological distress were assessed among 205 patients with newly diagnosed breast cancer by home interview 3 and 18 months after surgery. RESULTS. At 3 months, proportions of women with high distress reporting 0-1, 2-3, 4-5, and 6-15 stressful life events in the 5 years preceding diagnosis were 17%, 20%, 28%, and 37%, respectively (P = 0.006). High levels of psychological distress were present in 63.1% of women with a history of depression, compared with 14.3% of those with no such history (P = 0.0001). Associations of these factors with distress also were present 18 months after diagnosis. At 18 months only, distress was more frequent in women with regional disease (44%) than in those with localized disease (22%) (P = 0.006). Age, education, and marital status had little or no association with levels of psychological distress. CONCLUSIONS. Number of stressful life events before diagnosis and history of depression appear to be strong indicators of the risk of psychological distress and may be useful for identifying patients with breast cancer in need or more intense psychosocial support.     

Post-initiation treatment of Indole-3-carbinol did not suppress N-methyl-N-nitrosourea induced mammary carcinogenesis in rats.

The consumption of cruciferous vegetables (the Family of Cruciferae) such as cabbage, broccoli and Brussels sprouts has been shown to have cancer chemopreventive effects in humans and experimental animals. Indole-3-carbinol (I3C), one component of cruciferous vegetables, has been shown to exert cancer chemopreventive influence in liver, colon, and mammary tissue when given before or concurrent with exposure to a carcinogen. However in some reports, there has been evidence that consumption of I3C after carcinogen treatment might be associated with tumor promotion in some tissues. There have been no reports, to our knowledge, of post-initiation effects of I3C in the N-methyl-N-nitrosourea (MNU)-induced mammary tumor model in rats. Our studies were performed to examine this question. Ninety-six, 4-week-old female Sprague-Dawley rats were randomly divided into five groups. The animals of groups 1, 2 and 3 received an intraperitoneal injection of MNU at the age of 50 days. The animals of groups 4 and 5 were injected with saline only at the same time. Animals of groups 1 and 2 were given diet containing 100 ppm and 300 ppm I3C from week 1 until week 25 after MNU treatment. The animals of group 4 were given basal diet containing 300 ppm I3C without MNU treatment. All animals were killed at week 25. The incidences of mammary tumors in the groups 1, 2 and 3 were 95.8% (23/24), 83.3% (20/24) and 82.4% (28/34), respectively. The average number of tumors in the tumor bearing rats of the MNU and I3C 300 ppm group (group 2; 3.85+/-0.63) was higher than that in the MNU alone group (group 3; 2.46+/-0.31). These results represented that exposure to I3C after carcinogen treatment did not suppress development of mammary tumors.     

Structure-activity relationships for carcinogens with different modes of action.

The application of structure-activity concepts to the elucidation of the action of chemical carcinogens may proceed by two approaches: the hypothesis and the knowledge-based approaches. The former, exemplified by the 'structural alerts' devised by Ashby and associates, derives from the recognition of the electrophilic nature of carcinogens that damage DNA. The latter approach does not assume an a priori mechanism of action but derives information from the establishment of relationships between structural features and carcinogenicity. Indeed, the 'structural alerts' of Ashby et al. are recognized by such an approach; however, if structural features are associated with the activity of 'nongenotoxic' carcinogens, they would also be recognized by the knowledge-based approach. Obviously, the recognition of new (nonelectrophilic) structural features associated with carcinogenicity will lead to testable hypotheses.     

Influence of caffeine on development of benign and carcinomatous mammary gland tumors in female rats treated with the carcinogens 7,12-dimethylbenz(a)anthracene and N-methyl-N-nitrosourea

The effect of chronic caffeine consumption (500 mg/liter of drinking water) on the initiation and promotion stages of 7,12-dimethylbenz(a)anthracene (DMBA) (a low dose, 0.5 mg/100 g body weight, i.v.) and N-methyl-N-nitrosourea (MNU) (a standard dose, 2.5 mg/100 g body weight, i.v.) induced mammary gland tumorigenesis in female Sprague-Dawley rats was determined. In the initiation studies, caffeine was administered for 30 days prior to and for 3-4 days after carcinogen treatment (carcinogens administered at 55-57 days of age); in the promotion studies, caffeine was administered beginning 3-4 days after carcinogen treatment and until experiment termination (DMBA study and MNU study, 48 and 26 weeks after carcinogen treatment, respectively). In the DMBA study, there were 62-73 rats/group, in the MNU study, 40 rats/group. Eighty-nine % of the mammary tumors induced by DMBA were benign (adenomas, fibroadenomas, often with cystic secretory activity), 11% were carcinomas (intraductal and invasive); virtually all of the MNU-induced mammary tumors were carcinomas (approximately 99%). Caffeine consumption during the initiation stage in the DMBA-treated rats resulted in a significant decrease in the mean number of mammary carcinomas per rat (50% reduction, P less than 0.01) and mean number of benign mammary tumors per rat (28% reduction, P less than 0.05); caffeine consumption during the promotion stage significantly decreased the mean number of benign mammary tumors per rat (57% reduction, P less than 0.001) while not significantly influencing mammary carcinoma number. In contrast, caffeine consumption during either the initiation or promotion stages of MNU-treated rats did not significantly influence this tumorigenic process. The influence of caffeine on urinary and fecal excretion of tritiated DMBA and on rat mammary gland development at the time of carcinogen treatment also was determined. Slightly reduced levels of tritium in 24-h urinary samples were observed in caffeine-treated animals (P = 0.06). No significant effect of caffeine on 24- to 96-h fecal or 48- to 96-h urinary excretion of the isotope was observed. No apparent effect of caffeine on rat mammary gland development (number of ducts, degree of lobuloalveolar development) was observed. That caffeine significantly suppresses the initiation stage of DMBA-induced rat mammary gland tumorigenesis, while not influencing this stage when MNU is used as a carcinogen, suggests that caffeine acts via an alteration in carcinogen (DMBA) activation. The lack of a pronounced effect of caffeine on tritiated DMBA excretion, however, does cast some doubt on this mechanism.(ABSTRACT TRUNCATED AT 400 WORDS)     

DNA adduct formation and unscheduled DNA synthesis in rat esophagus in vivo after treatment with N-methyl-N-nitrosourea

An in vivo method for assessment of DNA adduct formation and unscheduled DNA synthesis (UDS) in the esophagus of rats was devised. Small ventral incisions were made in the neck and upper abdomen regions of 6 week old F344 rats and ligation of the esophagus with thread at the two extreme ends performed to make an esophageal pouch. For the DNA adduct formation study, a solution (0.5 ml) containing various concentrations of N-[3H]methyl-N-nitrosourea ([3H]MNU) was injected into the pouch. DNA binding levels were calculated from radioactivity of the isolated DNA and dose-dependent DNA adduct formation could be detected 2 h after the treatment with MNU. By HPLC analysis, both 7-methylguanine (7-mGua) and O6-methylguanine (O6-mGua) adducts were identified in the esophageal DNA, the ratio of 7-mGua/O6-mGua being 5.7-12:1. For UDS measurement, a solution containing MNU plus [3H]thymidine (200 microCi/ml) was similarly injected into the pouch. UDS was dose-dependently demonstrated as silver grains over the nuclei of the epithelial cells by autoradiography. The results thus showed that MNU, when injected into the esophageal lumen, can penetrate the surface mucosa, react with the epithelial cell DNA and induce DNA adduct formation and DNA repair synthesis dose-dependently.     

Unique patterns of gene expression changes in liver after treatment of mice for 2 weeks with different known carcinogens and non-carcinogens

Previously we demonstrated that the mouse liver tumor response to the non-genotoxic carcinogens oxazepam and Wyeth-14,643 involved more differences than similarities in changes in early gene expression. In this study we used quantitative real-time PCR and oligonucleotide microarray analysis to identify genes that were up- or down-regulated in mouse liver early after treatment with different known carcinogens, including oxazepam (125 and 2500 p.p.m.), o-nitrotoluene (1250 and 5000 p.p.m.) and methyleugenol (75 mg/kg/day), or the non-carcinogens p-nitrotoluene (5000 p.p.m.), eugenol (75 mg/kg/day) and acetaminophen (6000 p.p.m.). Starting at 6 weeks of age, mice were treated with the different compounds for 2 weeks in the diet, at which time the livers were collected. First, expression of 12 genes found previously to be altered in liver after 2 weeks treatment with oxazepam and/or Wyeth-14,643 was examined in livers from the various chemical treatment groups. These gene expression changes were confirmed for the livers from the oxazepam-treated mice in the present study, but were not good early markers for all the carcinogens in this study. In addition, expression of 20 842 genes was assessed by oligonucleotide microarray [n = 4 livers/group, 2 hybridizations/liver (with fluor reversals)] and the results were analyzed using the Rosetta Resolver System and GeneSpring software. The analyses revealed that several cancer-related genes, including Fhit, Wwox, Tsc-22 and Gadd45b, were induced or repressed in unique patterns for specific carcinogens and not altered by the non-carcinogens. The data indicate that even if the tumor response, including molecular alterations, is similar, such as for oxazepam and methyleugenol, early gene expression changes appear to be carcinogen specific and seem to involve apoptosis and cell cycle-related genes.     

VAB-6 as initial treatment of patients with advanced seminoma

Thirty patients with advanced seminoma were treated with VAB-6. Eighteen patients were previously untreated, eight had relapsed after radiation therapy, and four had persistent disease following chemotherapy and radiation therapy. Two patients had received prior high-dose cisplatin. Twenty-four (86%) of 28 evaluable patients achieved a complete remission. Four patients had relapsed. The median disease-free follow-up of patients achieving complete remission was 32+ months. VAB-6 is effective treatment for patients with advanced seminoma, and chemotherapy is recommended as the initial therapy in all patients with stage II seminoma with disease larger than 5 cm, extragonadal seminoma, and stage III seminoma.     

Induction of bladder cancer in rats by fractionated intravesicular doses of N-methyl-N-nitrosourea.

Experiments were conducted to determine the dose response of rat bladder urothelium to a range of different single and fractionated intravesicular doses of the carcinogen, N-methyl-N-nitrosourea (MNU). A dose-related response of bladder-tumour incidence to single graded doses of MNU was found, and a threshold does suitable for use of multistage carcinogenesis experiments was derived from these data. For any given total dose of MNU, the tumour incidence was greater if the MNU had been administered in several small fractions than if it had been administered in fewer larger ones. Extending the interval between doses did not reduce the tumour incidence. It is argued that these results support the multistage theory of carcinogenesis. The histopathology and cell-surface alterations which characterize the development of MNU-induced bladder cancer are described and the contribution of hyperplasia and calculi are discussed.     

Sequential effects of chemically different carcinogens,dimethylnitrosamine and 4-dimethylaminoazobenezene,on hepatocarcinogenesis in rats

Induction of liver tumors was studied in rats given carcinogens as follows: (1) N-nitrosodimethylamine (DMN) alone; (2) 4-dimethylaminoazobenzene (DAB) alone; (3) DMN followed by DAB; and (4) DAB followed by DMN. The main findings were: (1) DAB feeding for 0.62 to 2.5 months, followed by DMN feeding for 5 months, induced liver carcinomas more frequently than did DMN alone given for 5 to 10 months, or DAB alone given for 2.5 months. (2) Likewise, DMN feeding for 0.62 or 1.25 months, followed by DAB feeding for 5 months, induced liver carcinomas more frequently than did DMN alone given for 5 to 10 months or DAB alone given for 2.5 months. Interestingly, when DMN was given for a longer time (2.5 months), followed by DAB for 5 months, 50% of the induced liver tumors were non-epithelial. (3) Liver tumors were induced by sequential administration of the two carcinogens in doses that did not induce tumors when each carcinogen was given alone over a comparable time period. (4) The latent period for liver tumor development was shorter in groups that received the two carcinogens sequentially than in those that received DAB or DMN alone for comparable time periods.     

Inhibition of mammary tumorigenesis by a reduction of fat intake after carcinogen treatment in young versus adult rats

The present study demonstrates that a reduction of fat intake after dimethylbenz[alpha]anthracene (DMBA) administration to female Sprague--Dawley rats leads to an inhibition of mammary tumorigenesis. Animals were fed a 20% fat diet from weaning and were transferred to a 0.5% fat diet 0, 2, 4, and 6 weeks after carcinogen treatment. In rats given DMBA at 50 days of age, the following observations were obtained: (a) tumor incidence, as well as tumor yield, was decreased when the transfer to a low fat diet was initiated up to 4 weeks after DMBA; (b) regardless of fat intake, over 90% of tumors developed in all dietary groups were adenocarcinomas. This was in contrast to rats given DMBA at 150 days of age. In this case (a) a 50% reduction in tumor incidence was apparent when the low fat diet was introduced even 6 weeks after DMBA intubation; and (b) more benign lesions were found and an association between a reduced risk of carcinogenesis and a lower ratio of adenocarcinoma to fibroadenoma seems to exist. Thus, the data demonstrate that the rats were less vulnerable to delays in reduction of fat intake on subsequent inhibition of mammary tumorigenesis than if they were exposed to the carcinogen at an older age.     

Assay for mutagenicity of bile in Sprague-Dawley rats treated subcutaneously with intestinal carcinogens.

To investigate the mode of action of sc injected intestinal carcinogens, the mutagenicity assay of bile collected from noninbred Sprague-Dawley rats treated sc with carcinogens was conducted in the presence and absence of beta-glucuronidase. The bile samples from rats inoculated with 4-aminobiphenyl were mutagenic for Salmonella typhimurium TA100 only in the presence of beta-glucuronidase, whereas those from the 3,2'-dimethyl-4-aminobiphenyl-treated rats did not require the enzyme for mutagenicity toward strain TA100. On the contrary, the assays with S. typhimurium G46 and TA100 of bile from rats inoculated with 1,2-dimethylhydrazine, azoxymethane, or methylazoxymethanol acetate failed to reveal mutagenicity whether beta-glucuronidase was added or not, though these carcinogens were highly mutagenic for strain G46 in the Salmonella-microsome mutagenicity test and/or in the host-mediated assay.