Experimental and Monte Carlo investigation of mass attenuation coefficients of fission product isotopes in molecular precipitates.

Mass attenuation coefficients for molecular precipitates have been measured and compared to results using the EGS5 Monte Carlo computer code and results from an empirical formula. This study assesses the mass attenuation coefficients of isotopes that can be readily produced by thermal neutron activation of elements in a reactor and precipitated in molecular compounds. Good agreement exists between measured results and EGS5 simulated results. Results are within reasonable agreement with the empirical mass attenuation formula. These results are further compared to simulated fission product isotopes. This study suggests mass attenuation coefficients of molecular precipitates can be approximated using EGS5, especially in the instance of radioisotopes produced predominantly through uranium fission.

Effect of black tea on azoxymethane-induced colon cancer.

Two sets of experiments on the role of tea in azoxymethane (AOM) induced colon cancer were performed. The first test involved male F344 rats given 1.25% solutions of black tea beginning at 5 weeks of age and ending at 51 days of age. At 6 and 7 weeks of age, they received 15 mg/kg AOM and were held for 50 weeks. Another group received the AOM dosage at 6 and 7 weeks and were placed on the tea solutions 2 days after the last AOM dosage, at 51 days of age, and held for the 50-week period. The end point was the occurrence and multiplicity of colon cancer, classified as in situ, exophytic, invasive and Peyer's patch carcinomas. Tea failed to affect the incidence and multiplicity of colon cancers when given during or after the AOM administration, but tea after AOM increased the multiplicity of exophytic carcinomas. In a second series of tests, solutions of 0.6, 1.25, 1.75 or 2.5% tea were given, beginning 1 week prior to the two AOM doses and extending for 42 weeks. Also, one group received 1.25% tea and 1.85% whole milk. The incidence of exophytic or invasive colon cancer and tumor multiplicity were similar in all treatment groups, although the incidence of exophytic neoplasms was higher with 2.5% tea. Thus, chronic administration failed to significantly change the incidence and multiplicity of the AOM-induced colon cancers. These findings are accounted for by the underlying mechanism, namely the fact that tea solutions do not alter the amount of cytochrome P-4502E1 required for the metabolic activation of AOM.

The role of fat and calcium in the production of foci of aberrant crypts in the colon of rats fed 2-amino-1-methyl-6-phenylimidazo[4,5-b]-pyridine.

The modulation by dietary fat levels of intestine carcinogenesis is well documented. New developments suggest that calcium ions may also play a role. A rapid bioassay, the induction of foci of aberrant crypts in the colon, was used to explore the interaction between dietary fat and calcium. Male F344 rats 6 weeks of age were placed on diets containing 5 or 20% corn oil, and 0.04 or 0.32% calcium ion, as calcium lactate. Each dietary group was fed 400 ppm 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine (PhlP), and negative controls received the diets alone. A positive control group was given 2 mg N-nitrosomethylurea (NMU) intrarectally four times in a 2-week period. All rats were killed after 9 weeks. The intestinal tract was rinsed with Krebs-Ringer buffer. After staining a 6-cm segment of the descending colon and rectum with 0.2% methylene blue, foci of aberrant crypts were evaluated microscopically. With PhlP as a carcinogen, the rats on a high-fat, low-calcium level had more foci of aberrant crypts than animals on a low-fat level. With the higher calcium level, there were fewer foci and aberrant crypts, but the effect of fat was still significant. With NMU and a low-calcium level, the effect of fat level was evident. However, with the higher calcium intake, there were considerably more foci of aberrant crypts than on the low-calcium level, and the effect of the dietary fat level was not obvious.(ABSTRACT TRUNCATED AT 250 WORDS)

Genotoxicity and carcinogenicity in rats and mice of 2-amino-3,6-dihydro-3-methyl-7H-imidazolo[4,5-f]quinolin-7- one: an intestinal bacterial metabolite of 2-amino-3-methyl-3H-imidazo[4,5-f]quinoline.

BACKGROUND: Compounds formed on the surface of fried or grilled meat and fish may be associated with increased risk of colon cancer. Normal intestinal bacteria can convert one of these compounds, 2-amino-3-methyl-3H-imidazo[4,5-f]quinoline (IQ), to the 7-hydroxy metabolite, 2-amino-3,6-dihydro-3-methyl-7H-imidazolo[4,5-f]quinolin-7-o ne (7-OHIQ), a direct-acting mutagen. PURPOSE: We studied the genotoxicity and carcinogenicity of 7-OHIQ to determine if it is responsible for the colon-specific activity of IQ. METHODS: The effects of pure, synthetic 7-OHIQ on DNA were evaluated in the Ames Salmonella typhimurium TA98 test, with and without an induced rat liver S9 fraction, and in the Williams DNA repair test using freshly explanted rat hepatocytes. 7-OHIQ was also subjected to an in vivo bioassay for 21 months by long-term intrarectal infusion in male F344 rats, using IQ and N-nitrosomethylurea (NMU) given intrarectally as positive tumor-producing controls. The standard NIH-07 rodent diet was supplemented with 15% corn oil to maximize any effect of the infused materials on the colon. A parallel bioassay involved intraperitoneal injection of 7-OHIQ in newborn mice, followed by dietary administration from week 11 to week 67. Again, IQ and NMU were used as positive controls. RESULTS: We confirmed that 7-OHIQ is a direct-acting mutagen in the Ames test, with added S9 liver fraction giving higher mutagenicity. 7-OHIQ was negative in the Williams test, whereas IQ was positive. 7-OHIQ did not induce colon cancer in rats, and in the newborn mouse test it produced only a low incidence of liver neoplasms. CONCLUSIONS: 7-OHIQ is not genotoxic, for to be so classified it must be definitely positive in both the Ames and Williams tests; moreover, it is not carcinogenic, in marked contrast to IQ and NMU.

Comparative tumorigenicity in newborn mice of chrysene- and 5-alkylchrysene-1,2-diol-3,4-epoxides.

We determined the tumorigenicity in newborn mice of racemic anti-1,2-diol-3,4-epoxides of chrysene, 5-methylchrysene, 5-ethylchrysene and 5-propylchrysene. Among the four diol epoxides, only anti-5-methylchrysene-1,2-diol-3,4-epoxide was highly tumorigenic. It was 15-30 times more potent in induction of pulmonary tumors than the other compounds. The results demonstrate that molecular shape is critical in determining the tumorigenic activity of alkylchrysene diol epoxides. A methyl group in the same bay region as the epoxide ring leads to exceptional activity. This may be a consequence of DNA adduct conformation.

Effects of catechol on the induction of tumors in mouse skin by 7,8-dihydroxy-7,8-dihydrobenzo[a]pyrenes.

Catechol (1,2-dihydroxybenzene) is a major phenolic compound present in the co-carcinogenic fraction of cigarette tar. It has been shown to be a potent co-carcinogen with benzo[a]pyrene (BaP) in mouse skin. In this study we have examined the co-carcinogenic and co-initiating activities of catechol with racemic and enantiomeric 7,8-dihydroxy-7,8-dihydrobenzo[a]pyrenes (BaP-7,8-diols) in mouse skin. Similar to enhancement of BaP carcinogenesis, repeated concurrent applications of catechol and (+/-)-BaP-7,8-diol to mouse skin strongly enhanced (+/-)-BaP-7,8-diol tumor multiplicity and tumor incidence, and decreased latency. Co-application of catechol with the racemic or either of the enantiomers of BaP-7,8-diol in a two-stage initiation--promotion protocol increased the tumor initiating activity of racemic BaP-7,8-diol, similar to that of BaP, by approximately 50%, but had no statistically significant effect on the tumor initiating activity of the (+)- or (-)-enantiomers in mouse skin. Thus, catechol is as potent a co-carcinogen with (+/-)-BaP-7,8-diol as it is with BaP. However, as tested here catechol is a weak co-initiator when applied with (+/-)-BaP-7,8-diol or BaP.

Dietary beta-carotene in rat models of gastrointestinal cancer.

The effect of dietary beta-carotene (BC) was investigated in models of gastric and colonic carcinogenesis. Male Wistar rats were fed a diet with 0.4% BC during weaning, then 0.2% BC throughout. Cancer in the stomach and small intestine was induced by giving 80 mg/l N-methyl-N'-nitro-N-nitrosoguanidine (MNNG) in drinking water for 52 wk, but BC failed to affect carcinogenesis under these conditions, although the incidence of gastric adenocarcinoma was reduced slightly. Neoplastic and nonneoplastic lesions in the liver, skin, and pancreas were also present to a similar extent with BC feeding and without BC. Colorectal cancer was induced by six 2 mg intrarectal infusions of MNNG per rat over a 3-wk period, with the rats held another 22 wk without an inhibitory effect by BC. Thus, 0.2% dietary BC failed to influence significantly the development of neoplasia induced by a direct-acting carcinogen in the gastrointestinal tract.

Tumorigenic activity of non-alternant polynuclear aromatic hydrocarbons in newborn mice.

The tumorigenic activity of benzo[b]fluoranthene, benzo[j]fluoranthene, benzo[k]fluoranthene, and indeno-[1,2,3-cd]pyrene was evaluated in newborn CD-1 mice. The total doses of these non-alternant polycyclic aromatic hydrocarbons employed in this study ranged from 0.5 to 2.1 mumol per mouse. The results of this assay indicate that both benzo[b]fluoranthene and benzo[j]fluoranthene exhibit significant tumorigenic activity. In contrast to these results, neither benzo[k]fluoranthene nor indeno[1,2,3-cd]pyrene were tumorigenic under these assay conditions.

Methylated derivatives of pyrene and fluorene: evaluation of genotoxicity in the hepatocyte/DNA repair test and tumorigenic activity in newborn mice.

The genotoxicity of 1-methylpyrene, 1,6-dimethylpyrene, 1-methylfluorene, 9-methylfluorene, and 1,9-dimethylfluorene was evaluated in the hepatocyte primary culture/DNA repair test, and the tumorigenic potency of these compounds was tested by bioassay in newborn mice. With the exception of 1-methylfluorene, all of these methylated polycyclic aromatic hydrocarbons are known mutagens in Salmonella typhimurium. Both 1-methylpyrene and 1,6-dimethylpyrene induced unscheduled DNA synthesis in rat hepatocytes. However, only 1-methylpyrene was tumorigenic when administered to newborn male mice. None of the methylated fluorenes assayed in the hepatocyte primary culture/DNA repair test induced unscheduled DNA synthesis. While 1,9-dimethylfluorene exhibited weak tumorigenic activity when administered by subcutaneous injection to newborn mice, 1-methylfluorene and 9-methylfluorene were inactive.

Induction of oral cavity tumors in F344 rats by tobacco-specific nitrosamines and snuff.

The tumorigenic activities toward the oral cavity of snuff, its extracts, and two of its major nitrosamines, N'-nitrosonornicotine (NNN) and 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK) were evaluated in male F344 rats. In one protocol, groups of 21-30 rats were treated beginning at age 10 weeks by chronic application to the oral cavity for 131 weeks of either H2O, an H2O extract of snuff, an H2O extract of snuff enriched with ten times its indigenous concentration of NNN and NNK, or with NNN and NNK in H2O. The incidence of oral cavity tumors in the rats treated with NNN and NNK was 8 of 30, compared to 0 of 30 in controls (P less than 0.05). These results demonstrate that NNN and NNK can induce tumors locally in the oral cavity of F344 rats. Oral cavity tumors were also observed in 3 of 30 rats treated with snuff extract enriched with NNN and NNK, but not in the rats treated with snuff extract alone. In a second protocol, a test canal was surgically created in the lower lip of groups of 21-32 rats, and either snuff, H2O-extracted snuff, or snuff enriched with its own H2O extract was inserted in the test canal 5 times weekly for 116 weeks. A group of 10 control rats had surgery only. Among the 32 rats treated with snuff, 3 had oral cavity tumors; one was a squamous cell carcinoma originating in the test canal and invading the gingiva, one was a papilloma of the test canal, and one was a papilloma of the hard palate. Oral cavity tumors were also observed in 2 of 21 rats treated with H2O-extracted snuff and 1 of 32 rats treated with snuff enriched with its H2O extract. Oral tumors were not observed in control rats. The results of this study indicate that snuff and individual nitrosamines present in snuff can induce oral cavity tumors in F344 rats and support the epidemiological observations which indicate that snuff dipping causes oral cancer in man.

Chronic pulmonary inflammation modulates the fate of proteins administered by the respiratory tract.

The systemic appearance of radioiodinated proteins (125I-OA and 131I-HSA) administered via the respiratory route was studied in normal rabbits and in rabbits with BCG-induced chronic granulomatous pulmonary inflammation. The proteins were administered by i.t. injection into intact rabbits and into rabbits with tracheal cannulas or as an aerosol into isolated perfused lungs. The results showed that radioactivity appeared in the circulation as two fractions, one that was precipitables with 5% TCA and therefore protein-bound and one that was soluble in TCA. In both intact and tracheostomized animals, significantly more protein-;ound radioactive iodine was detected in the circulation of BCG-treated animals than in normal animals as early as 15 min after i.t. injection, and the differences persisted from 2 to 4 hr. However, in the isolated perfused lung, in which the only route for protein uptake into the circulation was the alveolocapillary barrier, only minimal differences in blood protein levels were observed as compared to normal BCG-inflamed lungs. This study suggests that chronic pulmonary inflammation promotes the absorption of i.t.-injected protein into the circulation, and that the route of enhanced uptake into blood is not the alveolocapillary membrane.

Effect of hypersensitivity on protein uptake across the air-blood barrier of isolated rabbit lungs.

In previous studies with isolated perfused rabbit lungs, we observed that human serum albumin (HSA) and ovalbumin, introduced into the isolated lungs as an aerosol, entered the pulmonary circulation antigenically intact. The "inhaled" proteins were also broken down in the lung. When lungs from animals immunized with one protein inhaled the two proteins simultaneously, absorption of intact antigen was specifically reduced, and there was a nonspecific increase in the appearance of metabolites of both proteins in the blood. In the present study, we investigated the antigen-specific and nonspecific effects of two types of hypersensitivity responses on protein absorption across the air-blood barrier of isolated rabbit lungs. In one group of lungs, an acute hypersensitivity response was induced by introducing HSA into the blood perfusing lungs from HSA-immunized rabbits. In another, the rabbits had been previously exposed to chronic HSA aerosol until their lungs exhibited a chronic immunologic inflammatory response. Lungs from both groups were insufflated simultaneously with HSA, and a nonspecific protein, ovalbumin. Lungs in which the acute anaphylactic response was induced showed no alteration in the absorption of either intact protein compared with HSA-immunized controls, but absorbed a somewhat larger quantity of breakdown products of the specific antigen. Lungs undergoing the chronic alveolar inflammation were more permeable to nonspecific protein than were noninflamed lungs. Despite the increased permeability to nonspecific protein, the absorption of antigen was blocked as effectively as in immune but noninflamed controls. In these chronically inflamed lungs, the absorption of antigen breakdown products was enhanced. The results indicate that both immunologic and inflammatory mechanisms may control the amounts of inhaled soluble proteins that reach the blood via the alveolocapillary barrier. Alterations in the absorption of inhaled proteins and their metabolites across the air-blood barrier during certain types of hypersensitivity responses may be of immunologic and pathologic significance.

An animal model of hypersensitivity pneumonitis in rabbits. Development of chronic pulmonary inflammation and cell-mediated hypersensitivity after repeated aerosol challenge.

Chronic pulmonary inflammation was produced in immunized rabbits by repeated aerosol exposure to soluble antigen. The pulmonary inflammatory response was correlated with the development of cell-mediated hypersensitivity in the lung as evaluated by migration-inhibition studies using bronchoalveolar cells. Such inflammation could be produced with either pigeon dropping extract, an etiologic agent of hypersensitivity pneumonitis, or with human gamma globulin. Development of the inflammatory response was immunospecific and could not be transferred to normal recipients with large quantities of immune serum. Collectively, these data suggest that the development of pulmonary inflammation was due to a cell-mediated immunologic reaction in the lung.

Immunologic block against antigen absorption from isolated perfused rabbit lungs.

In previous studies with isolated perfused rabbit lungs, we observed that inhaled human serum albumin (HSA) or ovalbumin (OA) entered the pulmonary circulation antigenically intact. The inhaled proteins were also metabolized in the lung. Immunization reduced the amount of intact protein and increased the amount of metabolites absorbed. In the present study, we have begun to characterize the immune mechanisms responsible for reduced antigen absorption. A humoral immune mechanism appeared to be involved because the phenomenon could be passively transferred to normal animals by administering immune serum either 18 hr or immediately before antigen inhalation. The reduction was also observed when lungs from immunized rabbits were perfused with normal rabbit blood, indicating that antibodies in both lung and blood may be involved. Experiments, in which lungs from immunized rabbits were simultaneously insufflated with the immunizing antigen and with a nonspecific protein, demonstrated that the block against antigen absorption was specific for the immunizing antigen and was not due to some antigen-induced nonspecific changes in lung physiology.

Absorption of inhaled antigen into the circulation of isolated lungs from normal and immunized rabbits.

The purpose of this study was to determine whether the absorption of inhaled antigen (Ag) across the pulmonary air-blood barrier of the isolated perfused lung can be modulated by immunologic mechanisms. Lungs from immunized or nonimmunized rabbits were removed, ventilated, and perfused with autochthonous blood. Radioiodinated Ag (human serum albumin or ovalbumin) was introduced as an aerosol into the isolated lung for 15 min and blood samples were taken over a 4-h period. The results showed that radioactivity fom inhaled Ag entered the perfusing blood as two fractions. One fraction was precipitable by 5% trichloroacetic acid or antiserum. The TCA-soluble fraction chromatographed differently from iodide and may have represented metabolites of the Ag. Immunization specifically reduced the amount of antigenically intact protein entering the blood. On the other hand, the metabolite reached higher concentrations in the blood of immunized lungs. We conclude that the alveolar capillary barrier of the normal rabbit lung could provide a significant route of entry for inhaled antigen into the systemic circulation and that immunization reduces absorption via this route and enhances pulmonary metabolism of the Ag.

Extraction and radioimmunoassay of urinary aldosterone in the rat.

Results from studies of the rat renin-angiotensin-aldosterone axis have been inconsistent and oftentimes conflicting among various laboratories and have led many investigators to regard the rat as a poor model with which to study this sytem. Many of these discrepancies have been shown to be due to the use of anesthetic agents in experimental protocols. Studies of unanesthesized animals indicate that the rat bears a remarkable resemblance to other mammalian species, but unfortunately the unanesthesized rat lends itself well only to acute studies, usually requiring killing the animal. Herein is described a method which permits the estimation of aldosterone excretory rates in intact, nonstressed, unanesthesized rats. The method is based upon the excretion and radioimmunoassay of urinary "acid-labile" or 3-oxo-conjugate of aldosterone. It is very sensitive, permitting the detection of less than 10 pg of aldosterone conjugate in extracted samples, and when compared to the double-isotope-dilution method, it is relatively inexpensive and much less tedious. Radioimmunoassays of rat urinary aldosterone excretion during 14.75 days of sodium depletion reflected a brisk increase in urinary excretion rate after day 2 and a concomitant reduction in sodium excretion that bears a remarkable resemblance to the excretory patterns described for man.