During incomplete combustion of organic matter there is formation of polycyclic aromatic hydrocarbons (PAH), which can react with oxides of nitrogen, with the generation of nitro-PAH's as a result, a reaction which is catalyzed by a low pH. 2-Nitrofluorene (NF), a marker for nitro-PAH, is metabolized in vivo via two different routes. After inhalation there is a formation of potent mutagenic metabolites, ON-NF's, which are distributed in the body. After oral administration, NF is reduced to the amine, a reaction mediated by the intestinal microflora, and further acetylated to 2-acetylaminofluorene (AAF), a potent carcinogen. Further ring-hydroxylation of AAF leads to detoxification and excretion. Induction of cytochrome P450c, d affects the metabolism in that more OH-NF's are formed. As a consequence, more mutagenic metabolites are found in the circulation. The liver excretes OH-NF's as, in terms of mutagenicity, glucuronide conjugates. When these conjugates are excreted via the bile, intestinal beta-glucuronidase can liberate direct-acting mutagens in the intestine. Thus, inhalation of NF can lead to formation of potent mutagens in the intestine. NF induces DNA-repair, in vivo, and is an initiator and a weak promotor, measured as formation of preneoplastic lesions in the liver. Risk estimates, by two different methods, indicate that nitro-PAH's extrapolated from the marker NF, can expose humans to a cancer risk at a nonneglectable level.

There is a falling longitudinal gradient of P-450IA1 along the length of the small intestine of rats fed a stock diet or a BNF-containing diet. In rats fed a special diet, based on purified ingredients, the gradient was abolished but could be restored by addition of betanaphthoflavone (BNF) to the diet. This indicates that the distribution of P-450IA1 in the rat small intestine is regulated by dietary components. Tape-section autoradiographic analysis showed that exposure to dietary BNF was an important determinant for the disposition of C-14-Trp-P-1. Following dietary BNF treatment, a pronounced retention of radioactivity was observed in the epithelium of the oral cavity, esophagus, forestomach and small intestine, but not in the glandular stomach. This was obvious but less striking in rats given BNF intraperitoneally, whereas no retention could be observed in the gastrointestinal tract of untreated rats.

Absorption, metabolism and DNA binding of 2-nitrofluorene (NF) was studied in isolated, perfused and ventilated rat lungs and in lung microsomal incubations. Comparisons were made between control animals and animals treated with beta-naphthoflavone (BNF), a 2,3,7,8-tetrachlorodibenzo(rho)dioxin (TCDD) receptor ligand and inducer of cytochrome P450IA1. Clearance of NF increased significantly in the isolated, perfused and ventilated lungs after BNF dosage, from 0.55 +/- 0.06 ml/min to 2.37 +/- 0.62 ml/min (P less than 0.05, n = 5-6). As a consequence of this, the mean residence time (MRT) for NF decreased when NF was dosed directly to the perfusion buffer, from 213 +/- 23 min (n = 6) to 48 +/- 9 min (n = 6), and after intratracheal dosage from 289 +/- 101 min (n = 5) to 135 +/- 72 min (n = 5). Irreversible binding of NF metabolites to DNA increased 2-fold after treatment with BNF when NF was dosed to the lung perfusion buffer. Treatment with BNF increased the rate of lung microsomal NF metabolism significantly, from 54 +/- 5 to 106 +/- 11 pmol/min/mg protein (P less than 0.05, n = 6-12). Formation of the monohydroxylated metabolite X-OHNF was inhibited in vitro by addition of alpha-naphthoflavone (50 microM), by 89 and 98% with lung microsomal fractions from control and BNF-treated rats respectively. In contrast, proadifen (50 microM) preferentially inhibited formation of 9-OHNF, by 42 and 33% in incubations with lung microsomal fractions from control and BNF-treated animals. Anti-P450IIB1-IgG inhibited formation of 9-OHNF by 96 and 45% with lung microsomes from control and BNF-treated rats respectively. Formation of X-OHNF was unaffected by addition of anti-P-450IIB1-IgG in both cases. These results show that both constitutive and inducible microsomal rat lung enzymes metabolize NF. A constitutive enzyme, most likely cytochrome P450IIB1, catalyzes metabolic attack on NF with high preference for the 9-position. A BNF-inducible microsomal enzyme, most likely cytochrome P450IA1, catalyzes hydroxylation of NF both in the 9-position and in other positions. Increased metabolic clearance, metabolism and DNA binding of NF after BNF treatment suggest that the level and specificity of cytochrome P450 isozymes may be important determinants for toxicity and availability of NF in the rat lung.

Northern and Western blot analyses, and analyses of microsomal metabolism of the carcinogen 2-nitrofluorene (NF) were conducted with the aim of studying age dependent cytochrome P-450b levels in the rat lung. The level of P-450b homologous mRNA and corresponding protein is very low in lungs from fetal and newborn rats. The levels then increase between 3 and 4 weeks of age, and reach adult levels at 6-8 weeks. No sex differences were detected with regard to lung P-450b mRNA levels or catalytical activities. Lung microsomal metabolism of NF increased in parallel with the accumulation of P-450b homologous mRNA and microsomal cytochrome P-450b protein concentration. Formation of the major metabolite, and potent mutagen, 9-hydroxy-2-nitrofluorene (9-OHNF) was significantly inhibited by addition of polyclonal anti-P-450b-IgG, and by addition of the inhibitor proadifen to incubations with lung microsomal protein. It is postulated that the observed, profound age-related differences in level and activity of lung cytochrome P-450b are likely to affect both availability and the ratio of metabolic detoxification and activation of chemical carcinogens deposited in the lung.

Dissolution kinetics of benzo(a)pyrene (B(a)P associated with urban air particulates were investigated in vitro and related to availability of the carcinogen in the isolated, perfused, and ventilated rat lung. Calculations of bioavailability and mean dissolution time in the lung produced results in concordance with experimental data. By convolution calculation, with in vitro desorption as input function and the disposition of B(a)P dosed intratracheally to the lung in an alveolar surfactant solution as reference, hypothetical areas under B(a)P buffer concentration vs. time curves (AUC) were calculated. AUC value for 1.5 micrograms of microcrystalline (MCr) B(a)P was 16 +/- 2 pmol/ml/min (N = 6), which was identical with the corresponding experimental value of 16 +/- 5 pmol/ml/min (N = 6), from lung perfusion experiments. For B(a)P adsorbed to urban air particulates (UAP-1), predicted and experimental AUC values did amount to 7 +/- 2 (N = 5) and 10 +/- 4 (N = 5) pmol/ml/min, respectively. Absolute bioavailability was calculated by deconvolution to 87% for B(a)P in alveolar surfactant. In vitro dissolution of B(a)P from urban air particulates (UAP-1 to -4) revealed a wide variation in desorption characteristics for different particulate samples. Dissolution parameters determined in vitro were positively related to mean absorption time and mean dissolution time in the lung, using three different preparations. The precision in predictions of availability of B(a)P from in vitro desorption rates, with MCr and UAP-1 representing the greatest differences observed, supports the hypothesis that desorption is the rate-limiting step during uptake of B(a)P in the lung.(ABSTRACT TRUNCATED AT 250 WORDS)

The metabolism of 2-nitrofluorene (NF), a model substance for nitrated polycyclic aromatic hydrocarbons, was studied in the isolated perfused rat lung and liver. NF has been identified in urban air and diesel exhaust and occurs in the gas, as well as in the particulate phase. Therefore, it is conceivable that the lung represents one point of entry of this compound into the body. The lung metabolizes NF to hydroxylated NFs, mainly 9-hydroxy-NF, independently of the route of administration (intravascular or intratracheal). After intratracheal administration, NF is rapidly excreted into the perfusate, indicating that other organs might be exposed to unmetabolized NF. The liver excretes NF metabolites as biliary glucuronides. Untreated bile is not mutagenic. However, after beta-glucuronidase treatment of bile, direct-acting mutagens were detected. The mutagenic metabolites in beta-glucuronidase-treated bile were the same as identified in the perfusate of the isolated lung. Since beta-glucuronidase is an enzyme found in the human intestinal microflora, inhalation of NF could result in the liberation of genotoxic metabolites in the colon.

Benzo[a]pyrene (B[a]P) adsorbed onto urban air particles (UAP) or in microcrystalline form (MCr) was administered intratracheally to the isolated perfused lung in doses of 100 and 1.5 micrograms. The appearance rate constant calculated for B[a]P release to the perfusate buffer was significantly lower for B[a]P administered adsorbed onto UAP (0.007 +/- 0.002 min-1) compared to the microcrystalline preparation (0.051 +/- 0.030 min-1). A classical two-compartmental model fitted well to the elimination of B[a]P from the perfusate buffer, after administration in solution to the buffer reservoir; C = 24 e-0.05t + 14 e-0.01t (pmol/ml). The concentration of polar metabolites in the perfusion buffer, at the end of experiments was approx. 9-fold higher for lungs administered the microcrystalline preparation compared to UAP at 1.5 microgram doses. At the 100 microgram dose level, the difference between preparations was only 2-fold, the data indicating that enzyme saturation might be important at the high dose level. With regard to the metabolite pattern, adsorption of B[a]P onto urban air particles caused a relative increase in the formation of B[a]P-9,10-dihydrodiol, whereas the relative formation rate for phenols was decreased. The absolute levels of B[a]P metabolites covalently bound to DNA was significantly higher in lungs given the MCr preparation compared to the UAP. When calculated as the amount metabolites bound, in relation to the total amount polar metabolites at the end of perfusion, however, the UAP preparation was significantly more efficient to enhance the production of DNA binding metabolites; 2.62 +/- 0.59 X 10(-5) vs. 1.33 +/- 0.21 X 10(-5) (pmol covalently DNA-bound metabolites/mg DNA/pmol metabolites formed). The results indicate that urban air particles may exert a cocarcinogenic effect with polynuclear aromatic hydrocarbons by increasing the pulmonary residence time for the carcinogenic hydrocarbon and/or alter the metabolite pattern in a way that enhances the covalent binding of metabolites to DNA.

Benzo(a)pyrene [B(a)P] was administered intratracheally to the isolated, perfused, and ventilated rat lung as a model compound for urban air polycyclic aromatic hydrocarbons. The compound was given in two forms, adsorbed onto urban air particles (UAP) and as microcrystals (MCr). The appearance of unmetabolized B(a)P in the perfusion buffer differed significantly between the dosage forms, UAP releasing B(a)P with an apparent first-order rate constant of 0.007 ± 0.002 min−1 and the MCr preparation, correspondingly by 0.051 ± 0.030 min−1. The elimination of circulating B(a)P, administered directly to the buffer, was observed to be readily described by the classical two compartment model: C = 245 × e−0.050xt − 14 × e−0.010xt pmol/mL. The production of polar metabolites, released to the perfusate at 150 min, was about five fold higher for B(a)P administered as MCr compared to UAP preparations, at 1.5-μg doses. At 100-μg doses, there was only a two fold difference in the metabolite production, indicating an enzyme saturation at high dose levels. The metabolite pattern was affected by UAP adsorption of B(a)P, producing a relative increase in the formation of B(a)P-9,10-dihydrodiol, whereas the phenol formation was decreased. The covalent binding of reactive metabolites to DNA also differed significantly between groups. Normalizing the total binding, to the metabolite production, the UAP-adsorbed B(a)P produced DNA adducts more efficiently than the MCr preparation; 2.62 (±0.59) × 10−5 versus 1.33 (±0.21) × 10−5 pmol metabolites bound per mg DNA and per total amount, pmol, metabolites formed. Results indicate a possible mechanism for the co-carcinogenic action of exposure for B(a)P and a carrier particle by an increased pulmonary retention and/or altered metabolite patterns enhancing the formation of reactive and DNA damaging metabolites.