Effects of induction and age-dependent enzyme expression on lung bioavailability, metabolism, and DNA binding of urban air particulate-absorbed benzo[a]pyrene, 2-nitrofluorene, and 3-amino-1,4-dimethyl-5H-pyridol-(4,3)-indole.

The effect of interactions between urban air particulates (UAP) and carcinogens on bioavailability, metabolism, and DNA binding was studied in the isolated perfused and ventilated rat lung. The availability of benzo[a]pyrene (B[a]P) varied from 29 to 60% after intratracheal doses of carcinogen particulates dissolving extremely slow and fast, respectively. Several cytochrome P450 enzyme (P450) inducers acting as 2,3,7,8-tetrachlorodibenzo[p]dioxin-receptor ligands have been identified in UAP extracts. beta-Naphthoflavone (BNF) was used to study how P450 induction alters the lung metabolism of carcinogens. Pretreatment increased the lung clearance for B[a]P 8-fold and for 2-nitrofluorene (2NF) by a factor of four from 0.55 +/- 0.06 ml/min to 2.37 +/- 0.62 ml/min. Studies with the intact lung and with isolated lung cells show that carcinogen metabolism and pharmacokinetics depend both on the route of exposure and dosage and on the distribution of specific enzymes. A cytochrome P450IIB1 enzyme was detected in lung epithelial cells where it catalyzes 9-hydroxylation of 2NF. This rat lung 2NF-9-hydroxylation capacity increases in parallel with the age dependent up-regulation of lung P450IIB1 expression. Both human and rat lung tissue have the capacity to form 9-hydroxy-2-nitrofluorene (9-OH-2NF) that is mutagenic. A BNF-inducible P450IA1 was detected in endothelial and alveolar type II cells. Consequently, aromatic hydroxylation dominated when 2NF was dosed directly into the lung circulation. Pretreatment of rats with BNF before intratracheal B[a]P dosage induced lung cytochrome P450IA1. The 7,8-dihydroxy-9,10-oxybenzo[a]pyrene-deoxyguanosine adduct and the total lung DNA adduct levels increased significantly from a peak level of 75 +/- 8 to 151 +/- 19 fmole/mg DNA in lungs from control and BNF pretreated rats, respectively. 3-Amino-1,4-dimethyl-5H-pyridol-(4,3)-indole (TRP-P1) is a potent mutagen and carcinogen identified in UAP extracts. Dietary BNF pretreatment of rats altered the [14C]TRP-P1 distribution as analyzed by whole-body autoradiography. An enhanced retention was observed in the small intestine, forestomach, esophagus, and lung. UAP catalyzes oxygen radical formation and deoxyguanosine-8-hydroxylation, which were inhibited when the UAP samples were extracted with organic solvents or when they were incubated in the presence of desferroxamine. We therefore postulate that a polycyclic aromatic hydrocarbon autooxidation pathway may be responsible for generation of hydrogen peroxide, which may be further converted to hydroxyl radicals through an iron-dependent reaction.

Influence of different levels of dietary casein on initiation of male rat liver carcinogenesis with a single dose of aflatoxin B1.

To analyze the influence of different levels of dietary casein on the initiation process, male Wistar rats, pair-fed on isocaloric diets containing 5, 15 or 40% casein were initiated with a single dose of aflatoxin B1, 28 days after the experimental start. From day 4 after initiation and until selection of initiated cells was started, 25 days later, rats were fed the 15% casein diet, providing an identical dietary background during the selection period. Promotion/selection of initiated cells was performed by the combined treatment with 0.02% 2-acetylaminofluorene in the 15% casein diet for 2 weeks and a two-thirds partial hepatectomy (PH) in the middle of this period. The number of enzyme-altered hepatic lesions per rat was shown to increase with increasing content of casein in the diet, both when liver sections were stained for gamma-glutamyltransferase and with immunohistochemical staining for the placental form of glutathione-S-transferase. Non-initiated rats fed the different levels of casein exhibited a very low number of foci. Livers were secured also from non-initiated rats at the same point of time as initiation was performed. Whereas no significant differences in the total microsomal content of cytochrome P450 were observed, a higher microsomal capacity to perform 16 alpha-hydroxylation of 4-androstene-3,17-dione was observed in preparations from rats fed 40% casein, when compared with rats receiving the 5% casein diet. The dietary protein content at the time of initiation did not affect the expression of the c-rasHa, c-myc or c-fos protooncogenes, either at initiation, on day 3, or at PH.

Effects of dietary and intraperitoneally administered beta-naphthoflavone on mutagenicity and tissue distribution of Trp-P-1 in the rat.

The effect of dietary beta-naphthoflavone (BNF) on tissue retention of 3-amino-1,4-dimethyl-5H-pyrido[4,3-b]indole (Trp-P-1) was studied in the rat. Female rats, 3 weeks old, were fed a BNF-containing diet for 3 days before being dosed orally or i.v. with 14C-labelled Trp-P-1. The rats were killed at 4, 24 or 48 h after dosage and subjected to tape-section autoradiography. The tissue localization of Trp-P-1-derived radioactivity was compared to that observed in untreated rats and in rats given BNF i.p. Ethoxyresorufin-O-deethylase (EROD) activity and mutagenicity of Trp-P-1 in the Ames test, using S9 prepared from forestomach, glandular stomach, small intestine, liver and lung, were used as in vitro assays to measure the degree of cytochrome P450IA1 and/or P450IA2 induction. Dietary BNF treatment caused a 30- to 40-fold increase in EROD activity in the small intestine, but only a 2-fold increase in the liver and the lung. These inter-organ differences were not observed after i.p. administration of BNF. The increase in mutagenicity of Trp-P-1 in the Ames test could be correlated to the increase in EROD activity. The autoradiographic data showed that the route of administration of BNF as well as of Trp-P-1 were important for the tissue localization of Trp-P-1. Dietary BNF treatment caused a pronounced retention of Trp-P-1-derived radioactivity in the epithelia of the small intestine, forestomach, oesophagus and the oral cavity, regardless of the administration route of Trp-P-1; a similar though less pronounced epithelial retention was observed after i.p. injection of BNF. A clear-cut boundary of accumulated radioactivity between the forestomach and the glandular stomach where the levels were almost non-detectable was observed in rats fed the BNF-containing diet. It is concluded that dietary inducers may be important determinants of metabolism and tissue distribution of toxic compounds.

Mutagen excretion and cytochrome P-450-dependent activity in germfree and conventional rats fed a diet containing fried meat.

To evaluate a possible role of the intestinal microflora in the metabolism of the highly mutagenic compounds formed in fried meat, conventional and germfree male AGUS rats were fed a semi-synthetic diet containing fried meat. Changes in mutagen excretion in urine and faeces over time were studied using the Ames Salmonella assay. The faecal and urinary extracts were separated by means of high-performance liquid chromatography (HPLC), and the mutagenicity of the collected fractions was determined. Cytochrome P-450 IA (IA1 and/or IA2) were detected by the use of antibodies with the Western blot technique, and the corresponding enzyme activities were measured in microsomes from the small intestine and the liver. A quantitative as well as qualitative difference in excretion of mutagens between germfree and conventional rats was observed. The total excreted of mutagenicity was significantly higher for the conventional than for the germfree rats, as a result of a higher faecal excretion of mutagens in the conventional animals. The HPLC separations of urinary and faecal extracts showed a different mutagenic metabolite pattern between the germfree and conventional rats. An increased activity of the cytochrome P-450-dependent enzyme ethoxyresorufin-O-deethylase was observed in the small intestine of conventional rats on the fried meat diet, whereas no effect of this diet was observed in the germfree rats. Similar results were obtained in immunoblotting experiments using a P-450 IA antiserum. The present study indicates that the excretion pattern and thus also the metabolism of compounds present in fried meat are affected by the germfree status.

Influence of fried meat and fiber on cytochrome P-450 mediated activity and excretion of mutagens in rats.

Fried meat was included in the diet of Sprague-Dawley rats during one week. Ingested and excreted amounts of mutagenic activity were determined daily by the use of the Ames' Salmonella/mammalian microsome test on extracts of the diet, urine and feces. In addition, the effect of the fried meat on ethoxyresorufin-O-deethylase activity in the small intestine and in the liver was measured. The results were compared to those from a control group of rats receiving boiled instead of fried meat as their source of protein. The diet containing boiled meat was not mutagenic and none of the samples from the control group, neither urine nor feces, contained any mutagenicity. The activity of ethoxyresorufin O-deethylase in the small intestine was increased by fried meat whereas this enzyme activity in the liver was unaffected. O-deethylation of ethoxyresorufin is catalyzed by cytochrome P-450 dependent enzyme(s) and induction of the enzyme activity might indicate an increased metabolic activation of premutagens and precarcinogens in the intestine. It is not yet known whether the mutagens excreted by the animals receiving the fried meat diet represent unmetabolized dietary mutagens or are formed as a result of metabolic activation of compounds in fried food. Dietary fibers i.e. wheat bran, pectin, cellulose or ViSiblin were included in the above-described diets and were shown to lower the mutagenic activity in urine and feces of the rats. Pectin significantly increased the hepatic ethoxyresorufin-O-deethylase and decreased the 16 alpha-hydroxylation of 4-androstene-3,17-dione.

Peripheral hormone levels in healthy subjects during controlled fasting.

The effect of one week of controlled fasting (31 of fluid containing 50 g of carbohydrate/day) upon the serum levels of hormones, sex hormone binding globulin, and albumin was studied in healthy subjects. Fasting caused decreased levels of prolactin and T3, no changes in the levels of TSH, FSH, LH, dehydroepiandrosterone, 4-androstene-3, 17-dione, total oestrone, and total testosterone, and increased levels of cortisol, dehydroepiandrosterone sulphate and albumin. A significant positive correlation was found between albumin and dehydroepiandrosterone sulphate. Fasting rapidly increased the levels of sex hormone binding globulin and decreased the percentage of free testosterone and the calculated free testosterone level in both sexes. A decreased metabolic clearance of certain steroids (cortisol, dehydroepiandrosterone sulphate) owing to an increased protein binding may be one of the endocrine consequences of fasting. An increased protein binding of testosterone may be outweighed by a decreased gonadal production, thus resulting in an unchanged total testosterone level. The increased sex hormone binding globulin level could not be explained by changes in gonadal and thyroid hormones.

Isolation of rat intestinal microsomes: partial characterization of mucosal cytochrome P-450.

A procedure is presented for the isolation of subcellular fractions from small intestinal mucosal cells in the rat. The mucosal cells were detached by a scraping procedure resulting in an almost complete harvest of all types of cells as judged by light microscopy. Homogenization using a Potter-Elvehjem Teflon-glass device at high speed with ensuing sonication was found to be necessary for complete disruption of the cells. The subcellular fractions obtained after differential centrifugation--10,000g pellet, 105,000g pellet (microsomal fraction), and supernatant--were characterized with respect to different marker enzymes. The highest yield of 7-ethoxyresorufin-O-deethylase and NADPH-cytochrome c reductase activity in the microsomal fraction was achieved after resuspension and recentrifugation of the 10,000g pellet. Addition of anti-P-450 beta-naphthoflavone (BNF)-B2 antibodies to the incubation mixture resulted in almost complete inhibition of the O-deethylation of 7-ethoxyresorufin whereas addition of anti-P-450 phenobarbital (PB)-B2 had no effect. The presence of BNF-inducible isozymes was demonstrated by the Western blotting technique not only in intestinal microsomes from BNF-treated rats, but also in microsomes from untreated rats. Anti-P-450 BNF-B2 was also used in the peroxidase-antiperoxidase method for studies on the localization of cytochrome P-450. No BNF-inducible cytochrome P-450 could be detected in untreated rats, whereas BNF treatment resulted in a general staining of the whole villus.