Adduct formation in hemoglobin of the newborn mouse exposed in utero to benzo[a]pyrene.

The administration of benzo[a]pyrene topically to pregnant mice during days 13-17 of gestation results in adduct formation in the hemoglobin of the mother and progeny. Thus, exposure to a total maternal body burden of 500 micrograms of benzo[a]pyrene during the last 5 days before delivery resulted in an average level of 6.35 (+/- 0.70 S.E.M.) pg of anti-diolepoxide metabolite covalently attached per mg of hemoglobin analyzed in the mother and 1.40 (+/- 0.23 S.E.M.) in the newborn animals. These data indicate that benzo[a]pyrene administered to the skin of the mother passed across the placental membrane, either as benzo[a]pyrene or some metabolite(s), and was present in the fetal tissue as the "ultimate" carcinogenic form (anti-diolepoxide metabolite) before binding to the hemoglobin. Concomitant adduct formation in the DNA of the skin with benzo[a]pyrene in the progeny was not observed and was probably due to the small amount of carcinogen applied to the mother. The data obtained, along with previously published results [Toxicology, 34 (1985) 211], suggest the suitability of hemoglobin as a molecular dosimeter for estimating carcinogenic risk to polycyclic aromatic hydrocarbons.

Detection of benzo(a)pyrene:DNA adducts in human white blood cells.

Metabolic activation of benzo(a)pyrene (BP) to its ultimate carcinogenic form, 7 beta, 8 alpha-diol-9 alpha, 10 alpha-benzo(a)pyrene epoxide (BPDE), and the binding of BPDE to DNA are important steps in BP carcinogenicity in experimental animals. Since people of certain occupations are exposed to high concentrations of BP, we have used enzyme-linked immunosorbent assay and ultrasensitive enzymatic radioimmunoassay to measure BPDE:DNA adducts in white blood cells from 2 of these occupational groups. Seven of 28 samples from roofers and 7 of 20 samples from foundry workers were positive for BPDE:DNA adducts (range, 2 to 120 fmol BPDE/50 micrograms DNA). In a group of nine volunteers without these industrial exposures to BP, the two positive DNA samples were from cigarette smokers. Control DNA obtained from human lymphocyte cell line RPMI 4265 was negative. These results indicate that the metabolic activation of BP and formation of BPDE:DNA adducts occurs in humans.

Ubiquitous binding of benzo[a]pyrene metabolites to DNA and protein in tissues of the mouse and rabbit.

The in vivo formation of benzo[alpha]pyrene (BP) metabolite-DNA adducts in several tissues of mice and rabbits was examined. Included were tissues with widely divergent xenobiotic metabolizing capabilities such as liver and brain. The major adduct identified in each tissue was the (+)-7 beta,8 alpha-dihydroxy-9 alpha,10 alpha-epoxy-7,8,9,10-tetrahydro-BP (BPDEI)-deoxyguanosine adduct. A 7 beta,8 alpha-dihydroxy-9 beta,10 beta-epoxy-7,8,9,10-tetrahydro-BP (BPDEII)-deoxyguanosine adduct, a (-)-BPDEI-deoxyguanosine adduct and an unidentified adduct were also observed. These adducts were present in all of the tissues of the mice and in the lungs of the rabbits; only BPDEI and BPDEII were seen in the rest of the rabbit tissues. In all of the tissues studied, the DNA adduct levels were unexpectedly similar. For example, the BPDEI-DNA adduct levels in muscle and brain of mice were approx. 50% of those in lung and liver at each oral BP dose examined. After an i.v. dose of BP in rabbits, the BPDEI adduct levels in lung were three times those in brain or liver and twice those in muscle. The binding of BP metabolites to protein was also determined in these tissues. The tissue-to-tissue variation in protein binding levels of BP metabolites was greater than that for BPDEI-DNA adducts. There are several possible explanations for the in vivo binding of BP metabolites to DNA and protein of various tissues. First, oxidative metabolism of BP in each of the examined tissues might account for the observed binding. Second, reactive metabolites could be formed in tissues such as liver and lung and be transported to cells in tissues such as muscle and brain where they bind to DNA and protein. In any case, the tissue-to-tissue variations in protein and DNA binding of BP-derived radioactivity do not correlate with differences in cytochrome P-450 activity.

Hydrocarbon transport in chylomicrons and high-density lipoproteins in rat.

A lipoprotein system is described that transports gut hydrocarbons of low polarity in chylomicrons of intestinal lymph and plasma to plasma high density lipoproteins (HDL) in rat. Four highly lipophilic aryl and alkyl hydrocarbons [benzo(alpha)pyrene; 1,1,1-trichloro-2,2-bis(p-chlorophenol)ethane (DDT), hexadecane and octadecane] were selected to give a graded range of polarity. Chylomicrons were labeled doubly with radioisotopes in triacylglycerol and a single hydrocarbon by feeding [3H]-glycerol and [14C]hydrocarbon. All hydrocarbons were transported in the triacylglycerol oil phase of chylomicrons. Injected chylomicron triacylglycerol and 3 of 4 hydrocarbons were cleared simultaneously from plasma consistent with lipoprotein-lipase dependent hydrocarbon clearance but DDT was cleared more rapidly. HDL was the major plasma acceptor of all labelled hydrocarbons. Plasma chemical fluxes were measured for octadecane and DDT and both showed net fluxes from chylomicrons to HDL. HDL selectively concentrated chylomicron hydrocarbons from chylomicron triacylglycerol. Lipoprotein lipase stimulation by intravenous heparin significantly increased transfer of alkanes from chylomicrons to HDL. These results indicate that (a) chylomicrons transport gut-derived hydrocarbons with a wide range of structure and polarity as triacylglycerol solutes; (b) HDL are a major plasma acceptor of all these hydrocarbons, demonstrating both selective solute uptake from triacylglycerol and net chemical uptake for the 2 hydrocarbons studied and (c) efflux of these chylomicron hydrocarbons from plasma and into HDL is regulated partly by hydrolysis of chylomicron triacylglycerol.

Factors affecting plasma benzo[a]pyrene levels in environmental studies.

Benzo[a]pyrene (BaP) is a useful indicator of exposure to polycyclic aromatic hydrocarbons (PAHs), airborne carcinogenic compounds. Radioimmunoassay was used to test for plasma BaP differences in 61 subjects divided into three groups based on geographic-demographic locale: urban-industrial, urban-residential, and outer suburban. The results showed that the urban-industrial area participants had a significantly higher mean plasma BaP level than did the outer suburban subjects. The urban-residential subjects did not have a significantly different mean plasma benzo[a]pyrene level from either of the other two groups. Obesity, as measured by Quetelet's index, was found to have a significant correlation with BaP levels. These results indicate that radioimmunoassay of plasma for BaP may be used successfully to judge environmental exposure to PAHs, provided physiological considerations such as obesity are taken into account.

Total body clearance of circulating benzo(a)pyrene in conscious rats: effect of pretreatment with 3-methylcholanthrene and the role of liver and lung.

The metabolic clearance of benzo(a)pyrene [B(a)P] was studied in conscious control and 3-methylcholanthrene (3-MC)-pretreated rats. The roles of liver and lung in total body B(a)P clearance were estimated in vivo in 3-MC-treated rats. During a 5-hr period, blood B(a)P concentration decreased rapidly in a biphasic manner. 3-MC pretreatment did not significantly affect the rate constants of B(a)P elimination, but the apparent volume of distribution increased, resulting in an increase in total body B(a)P clearance from 15 +/- 1 to 48 +/- 6 ml/min. Five hours after injection, B(a)P concentrations in most organs were lower in 3-MC-pretreated rats; however, lung B(a)P concentration was greater. In the 3-MC-pretreated rats, first-pass extraction of B(a)P by lung and liver was determined from comparison of areas under arterial blood B(a)P concentration vs. time curves after i.a., i.v. and intrahepatic portal venous administration. Liver had a first-pass extraction of 20% whereas that by lung was 33%. Organ B(a)P concentrations were correlated with the route of B(a)P administration. The results of this study suggest that 1) 3-MC pretreatment increases the apparent volume of B(a)P distribution in vivo, 2) liver and lung of 3-MC-pretreated rats are about equal in their ability to extract circulating B(a)P in vivo and 3) 3-MC pretreatment enhances total body B(a)P clearance by increasing extrahepatic B(a)P elimination.

Aromatic hydrocarbon metabolites in fish: automated extraction and high-performance liquid chromatographic separation into conjugate and non-conjugate fractions.

An automated extractor-concentrator was used to extract metabolites of naphthalene, 2,6-dimethylnaphthalene, and benzo[a]pyrene from serum, bile and liver homogenate of rainbow trout (Salmo gairdneri). The extracts were analyzed by reversed-phase high-performance liquid chromatography (HPLC) with fluorescence detection. Recoveries of naphthalene and 2,6-dimethylnaphthalene metabolites from all matrices were generally greater than 90%; however, the recoveries of benzo[a]pyrene metabolites from serum ranged from 37-99%. In addition, conjugated metabolites of polycyclic aromatic hydrocarbons (PAHs) were separated from non-conjugated metabolites and parent PAHs by using two diol columns with normal-phase HPLC. The extraction and separation techniques were also applied to isolate metabolites in samples from fish fed 2,6-dimethylnaphthalene.

Influence of sulfur dioxide on metabolism and distribution of benzo[a]pyrene in isolated perfused rabbit lung.

An isolated perfused lung [IPL) preparation was used to investigate the effects of SO2 (1-2 ppm) on the metabolism of benzo[a]pyrene (BaP), a ubiquitous potent carcinogen that has been associated with the increased incidence of a human bronchiogenic carcinoma in occupational and urban populations. [14C]BaP, with and without crude air particulate (CAP), was administered intratracheally to the IPL in conjunction with SO2 or after pretreatment of the whole animal with SO2. Metabolites were isolated from serial blood samples up to 3 h after the administration of [14C]BaP to the IPL. Metabolites were also isolated from lung tissue, washout fluid, macrophage, and trachea and bronchi at the end of the perfusion at 180 min. Patterns of BaP metabolites were determined by thin-layer and high-performance liquid chromatography and scintillation counting. SO2 given in conjunction with BaP on the IPL or given to the whole animal followed by BaP on the IPL, in comparison with BaP only on the IPL, resulted in a twofold increase in the total rate of appearance of metabolites of BaP in the blood with changes in the metabolic pattern. SO2 given in conjunction with BaP and CAP on the IPL, in comparison with BaP plus SO2 on the IPL, resulted in a threefold decrease in the total rate of appearance of metabolites of BaP in the blood with changes in the metabolic pattern.

The uptake and release of benzo[a]pyrene and benzo[e]pyrene in vitro by Syrian hamster embryo cells as a function of serum concentration.

A quantitative study on the in vitro uptake of benzo[a]pyrene (B[a]P) and benzo[e]pyrene (B[e]P) by Syrian hamster embryo cells and the induction of sister chromatid exchange (SCE) has been carried out. The amounts of B[a]P and B[e]P taken up by the cells decreases as does the induction of SCEs by B[a]P when the concentration of serum in the culture medium increases. It appears that serum prevents (B[a]P or B[e]P uptake. We have observed no significant differences between the two hydrocarbons regarding uptake by cells; chromatographic results show however that B[a]P is metabolized by these cells, while B[e]P is not. Our results suggest that serum inhibits B[a]P and B[e]P uptake and hence decreases the number of SCEs.

Serum and tissue distribution of benzol[a]pyrene from intravenously injected chylomicrons in rat in vivo.

Rats were injected intravenously with chylomicrons (CM) containing benzo[a]pyrene (B[a]P), a carcinogenic aromatic hydrocarbon. The disappearance of B[a]P paralleled the removal of CM, both having a rapid initial decay and a secondary slow decay. After 5 min, at the end of the rapid phase, blood contained 19% of the total injected B[a]P, with 6% in blood cells and 13% in serum. At 60 min, serum contained 5% and blood cells 6% of the total B[a]P. During the slow phase, further distribution of B[a]P within serum albumin and various lipoproteins occurred, most of the label being in low density and very low density lipoproteins. Highest tissue specific activities of [3H]B[a]P were in the lung, liver and kidney tissues. These results suggest that in the rat, distribution of ingested polycyclic aromatic hydrocarbons in vivo tends to be primarily determined by the catabolism of chylomicrons.

Pancreatic metabolism of benzo(a)pyrene in vitro and in vivo in the Long-Evans rat.

The in vitro metabolism of benzo(a)pyrene was compared between pancreas and liver with and without pretreatment. There was no significant difference in metabolic pathways between the two tissues. Evidence for induction of the metabolism was evident following 3-methyl-cholanthrene pretreatment. Temporal studies demonstrated rapid elimination of BP from the tissues within the first 5 hours with possible recirculation of metabolites 22 hours after injection of the carcinogen. There was significant binding of BP to DNA in pancreas and liver at 5 hours.

High-pressure liquid chromatographic analysis of benzo(a)pyrene metabolism by human lymphocytes from donors of different aryl hydrocarbon hydroxylase inducibility and antipyrine half-lives.

With high-pressure liquid chromatography (HPLC), lymphocytes from six human donors were evaluated for their ability to metabolize benzo(a)pyrene (BP). Donors whose aryl hydrocarbon hydroxylase (AHH) inducibility ratios ranged from 2.4 to 4.6 and whose antipyrine plasma half-lives ranged from 8 to 17 hr were examined. The BP metabolites identified were: 7,8-dihydrodiol, quinones, and 9-hydroxy and 3-hydroxy phenols. HPLC profiles of BP metabolites elaborated by uninduced (control) and benz(a)anthracene-induced lymphocytes were qualitatively similar among the six donors. A good correlation (r = 0.79) was found between known AHH inducibility ratios for the donors, as determined by the conventional fluorometric AHH assay, and induction of BP phenol production quantitated from HPLC data. HPLC results also indicated that the induction of benzo(a)pyrene-7,8-dihydrodiol, the proposed proximate carcinogenic form of BP, did not parallel BP phenol induction. Furthermore, the data also indicated a good negative correlation between AHH inducibility and the measurements of plasma antipyrine or urinary 4-hydroxyantipyrine half-lives (r = -0.88 or -0.91), respectively.

Absorption and metabolism of naphthalene and benzo(a)pyrene in the rat jejunum in situ.

Naphthalene or benzo(a)pyrene (100 nmol) was instilled into the closed rat intestinal loop in situ and the appearance of the free compound and its metabolites was determined in portal blood. Naphthalene appeared mostly unchanged in blood whereas benzo(a)pyrene was extensively metabolized by mucosal cells. The results suggest that absorption and metabolism are competing processes in the gut.