Recombinant aryl hydrocarbon receptors for bioassay of aryl hydrocarbon receptor ligands in transgenic tobacco plants.

Dioxin residues widely contaminate soil and agricultural products at low concentrations and may accumulate in organisms at the top of food chains owing to their physicochemical properties. In this study, we have developed novel, dioxin-inducible, reporter gene expression systems regulated by recombinant aryl hydrocarbon receptors (AhRs). The recombinant AhRs, referred to as XDVs, consist of the DNA-binding domain of the bacterial repressor protein LexA, a 90-kDa heat shock protein- and ligand-binding regulatory domain from mouse AhR, and the transactivation domain of herpes simplex virus regulatory protein VP16. Transgenic tobacco plants carrying XDVs absorb various AhR ligands, including 3-methylcholanthrene, beta-naphthoflavone and indigo from solid medium and vermiculite, and show dose- and time-dependent expression of the beta-glucuronidase reporter gene. The results clearly suggest that XDVs are functional transcription factors that respond to AhR ligands, and that the XDV-mediated reporter gene expression system is applicable to bioassays for dioxin residues in the environment.

Gas chromatographic assay of epoxide hydrase activity with 3-methylcholanthrene-11,12-oxide.

Epoxide hydrase has been measured in rat tissue with 3-methylcholanthrene-11,12-oxide as substrate; diol formation was assayed by gas chromatographic separation of the trimethylsilylated derivative of trans-11,12-dlhydro-11,- 12-dihydroxy-3-methylcholanthrene from the corresponding derivative of the 11 (or 12)-hydroxy-3-methylcholanthrene on 3 percent OV-17, which is formed from the 11,12-oxide during the derivatization. The polycyclic hydrocarbons were extracted initially from the incubation mixture with ethyl acetate. The assay is simple, inexpensive, and sensitive.

3-Methylcholanthrene and other aryl hydrocarbon receptor agonists directly activate estrogen receptor alpha.

3-Methylcholanthrene (3MC) is an aryl hydrocarbon receptor (AhR) agonist, and it has been reported that 3MC induces estrogenic activity through AhR-estrogen receptor alpha (ER alpha) interactions. In this study, we used 3MC and 3,3',4,4',5-pentachlorobiphenyl (PCB) as prototypical AhR ligands, and both compounds activated estrogen-responsive reporter genes/gene products (cathepsin D) in MCF-7 breast cancer cells. The estrogenic responses induced by these AhR ligands were inhibited by the antiestrogen ICI 182780 and by the transfection of a small inhibitory RNA for ER alpha but were not affected by the small inhibitory RNA for AhR. These results suggest that 3MC and PCB directly activate ER alpha, and this was confirmed in a competitive ER alpha binding assay and in a fluorescence resonance energy transfer experiment in which PCB and 3MC induced CFP-ER alpha/YFP-ER alpha interactions. In a chromatin immunoprecipitation assay, PCB and 3MC enhanced ER alpha (but not AhR) association with the estrogen-responsive region of the pS2 gene promoter. Moreover, in AhR knockout mice, 3MC increased uterine weights and induced expression of cyclin D1 mRNA levels. These results show that PCB and 3MC directly activate ER alpha-dependent transactivation and extend the number of ligands that activate both AhR and ER alpha.

Activation of the dioxin/aryl hydrocarbon receptor (AhR) modulates cell plasticity through a JNK-dependent mechanism.

Environmental chemicals such as dioxin adversely affect immune, neurological and reproductive functions and have been implicated in cancer development. However, the mechanisms responsible for dioxin toxicity are still poorly understood. Here, we show that dioxin and related pollutants trigger a marked morphological change in epithelial cells that remodel their cytoskeleton to increase interaction with extra cellular matrix while loosening cell-cell contacts. Furthermore, dioxin-treated cells show increased motility. These dioxin-mediated effects are mimicked by constitutive expression and activation of the intracellular dioxin receptor (aryl hydrocarbon receptor (AhR)). They correlate with activation of the Jun NH2-terminal kinase (JNK) and are reverted by treatment with a JNK inhibitor. Dioxin-induced effects occur 48 h post-treatment initiation, a time scale, which argues for a genomic effect of the AhR, linked to induction of target genes. This novel Ahr action on cell plasticity points to a role in cancer progression.

Activation of p53 as a causal step for atherosclerosis induced by polycyclic aromatic hydrocarbons.

This study was performed to prove our hypothesis that the metabolite(s) of polycyclic aromatic hydrocarbons (PAHs) caused the activation or phosphorylation of p53 via DNA damage to suppress the liver X receptor (LXR)-mediated signal transductions as a probably more direct mechanism. We found that LXR-mediated trans-activation was inhibited by 3-methylchoranthrene (MC) and doxorubicin (Dox) in HepG2 cells carrying wild-type p53, but not in Hep3B cells possessing mutant p53. The exogenous expression of wild-type p53 suppressed the LXR-mediated trans-activation in Hep3B cells. The expression of mRNA for ATP binding cassette A1 was suppressed by MC and Dox in HepG2 cells. The protein expression of retinoid X receptor (RXR), a partner of LXR to form a heterodimer, was suppressed by MC and Dox in HepG2 cells.

Environmental prognostics: an integrated model supporting lysosomal stress responses as predictive biomarkers of animal health status.

The potential prognostic use of lysosomal reactions to environmental pollutants is explored in relation to predicting animal health in marine mussels, based on diagnostic biomarker data. Cellular lysosomes are already known to accumulate many metals and organic xenobiotics and the lysosomal accumulation of the carcinogenic polycyclic aromatic hydrocarbon 3-methylcholanthrene (3-MC) is demonstrated here in the hepatopancreatic digestive cells and ovarian oocytes of the blue mussel. Lysosomal membrane integrity or stability appears to be a generic indicator of cellular well-being in eukaryotes; and in bivalve molluscs it is correlated with total oxygen and nitrogen radical scavenging capacity (TOSC), protein synthesis, scope for growth and larval viability; and inversely correlated with DNA damage (micronuclei), as well as lysosomal swelling (volume density), lipidosis and lipofuscinosis, which are all characteristic of failed or incomplete autophagy. Integration of multiple biomarker data is achieved using multivariate statistics and then mapped onto "health status space" by using lysosomal membrane stability as a measure of cellular well-being. This is viewed as a crucial step towards the derivation of explanatory frameworks for prediction of pollutant impact on animal health; and has facilitated the development of a conceptual mechanistic model linking lysosomal damage and autophagic dysfunction with injury to cells, tissues and the whole animal. This model has also complemented the creation and use of a cell-based bioenergetic computational model of molluscan hepatopancreatic cells that simulates lysosomal and cellular reactions to pollutants. More speculatively, the use of coupled empirical measurements of biomarker reactions and modelling is proposed as a practical approach to the development of an operational toolbox for predicting the health of the environment.

The uptake and secretion of 3-methylcholanthrene by the prostate glands of the rat and dog.

In anesthetized rats in which prostatic fluid was collected over a 2-hour interval from 24 to 26 hours after a single ip dose of 5 mg [6-14C]3-methylcholanthrene ([14C]MCA)/kg, radioactivity was found in the fluid at levels only slightly less than those in plasma; at 26 hours after treatment, levels of radioactivity within the prostate were higher than those in prostatic fluid or plasma. When unanesthetized dogs with surgically prepared prostatic fistulas were given a single ip dose of 0.5 mg [14C]MCA/kg and when serial prostatic fluid and plasma samples were collected over the ensuing 50 hours (2 dogs) or 212 hours (1 dog), radioactivity appeared in the prostatic fluid at levels initially greater than those in plasma and then fell progressively with time to less than those of plasma. At 50 hours after treatment, radioactivity was recovered from the prostate glands of 2 dogs with fistula and 2 dogs without fistula at levels of about one-fourth those of plasma. Thus it was found that [14C]MCA and/or its metabolites entered the prostate glands and prostatic fluids of the rat and dog.

Chemoprotective effect of progesterone on carcinoma formation in mice and rats.

The protective effect of progesterone on the development of chemically induced carcinomas (squamous cell carcinomas in mice and basal cell carcinomas in rats) by 3-methylcholanthrene [(MCA) CAS: 56-49-5] was studied. Progesterone administration decreased the average number, size, and weight of carcinomas by 45-50% as compared to those of tumors treated with MCA alone at any time interval. DNA radioactivity and autoradiographic studies with the use of [3H]thymidine showed an inhibition of DNA synthesis in the neoplastic cell nuclei following a concomitant administration of progesterone and MCA (18.4%) as compared to the DNA synthesis following administration of MCA alone (35.0%). Electron microscopic and cytologic observations revealed salient ultrastructural findings following progesterone administration, with advanced cytolysis, tumefied mitochondria, large populations of secondary lysosomes, and autophagic formations; also, cell differentiation tended to be of a glandular-adenomatoid type following progesterone and MCA administration as compared to the characteristic squamous cell and basal cell carcinomas after treatment with MCA alone. In addition, scanning electron microscopic observations revealed advanced cytolytic areas with several disintegrated neoplastic cells and cell debris intermingled with red blood cells, following progesterone and MCA administration. The present findings demonstrate that progesterone in pharmacologic doses exerts important chemoprotective effects on carcinoma formation, possibly by interfering with MCA metabolism and inhibiting DNA synthesis in the epidermal neoplastic cells, and thus plays an important role in tumorigenesis.

Enhancement of 5-iododeoxyuridine-induced endogenous C-type virus activation by polycyclic hydrocarbons: apparent lack of parallelism between enhancement and carcinogenicity.

When mouse MLg cells were treated with 3-methylcholanthrene or 7,12-dimethylbenz[alpha]anthracene in the presence of microsomal enzymes and NADPH after 5-iododeoxyuridine (IUDR) treatment, the induction rate of the endogenous C-type virus was increased fivefold to sixfold in comparison with the culture treated with IUDR only. In this reaction, both the microsomal enzymes and NADPH were indispensable. 7,8-Benzoflavone, an inhibitor of the metabolism of hydrocarbons in hamster embryo cultures, inhibited the reaction. For detecting the enhancing activity, the concentration of IUDR for the pretreatment, the concentration of the test products, and the duration of the treatment with the products were important factors. In screening 30 polycyclic hydrocarbons, we were unable to detect a correlation between the in vivo carcinogenicity in the skin and the enhancing activity in the conditions tested.

Persistent binding of 3-methylcholanthrene to mouse lung DNA and its correlation with susceptibility to pulmonary neoplasia.

A/J, C3H/HeJ, DBA/2J, and C57BL/6J mice have different susceptibilities to polycyclic aromatic hydrocarbon-induced pulmonary neoplasia, whereas the livers from these animals are uniformly resistant to the carcinogenic actions of these substances. After i.v. injection of [3H]-3-methylcholanthrene, radiolabel was detected in the DNA of both lung and liver of all these strains. The DNA was digested to deoxyribonucleosides and chromatographed on Sephadex LH-20. The amounts of nucleoside-bound adducts detected varied markedly with the different tissues and strains. These adducts were undetectable in liver DNA by 28 days. Although all lung preparations showed some reduction in adducts by 28 days, the amounts in A/J lung were always highest; this correlated with its high susceptibility to neoplastic transformation. In all preparations, radioactivity eluted from Sephadex LH-20 with the column void volume or with underivatized nucleosides. Tissue, but not strain differences were observed in these chromatographic profiles. The predominance of these early-eluting peaks in liver, rather than lung, suggests that they may represent noncarcinogenic lesions. This radiolabeled material remains uncharacterized, but some possibilities are discussed.

The isolation and characterization of polycyclic hydrocarbon-binding proteins from mouse liver and skin cytosols.

The major protein to which metabolites of methylcholanthrene are covalently bound has been purified from C3H mouse liver cytosol. Its properties are identical to the mouse skin h-protein, which may be primary arget of carcinogenic hydrocarbon metabolites during transformation to caner. It has a molecular wight of 44,000, consists of 2 subunits o- M.W. 20,000, has an isoelectric point (pI) of 8.05 to 8.6, and a sedimentation coefficient of 3.6 S. These physical properties are rather similar to those of ligandin, a hepatic protein that binds carcinogen metabolites, steroid anionic metabolites, bilirubin, and exogenous organic anions, but not to those of the rat liver azo dye carcinogen binding 'slow h-2-5S' protein. The h-protein and ligandin consistently give different pl values. Two minor basic proteins (molecular weights around 44,000 each), to whcih methylcholanthrene metabolites are convalently bound, have been separated from the h-protein by carboxymethyl-cellulose chromatography. Prelininary results indicate that these 2 minor proteins are related to ligandin. A protein to which methylcholanthrene is noncovalently bound was also identified in the acidic fraction of the mouse liver and skin sytosols and has been partially purified and characterized. It has a molecular weight of 60,000, a pl of 5.0, and a sedimentation coefficient of 4.5S.

Correlation of induction of aryl hydrocarbon hydroxylase in cultured rat hepatocytes with saturable high-affinity binding of 3-methylcholanthrene to a 4S cytoplasmic protein.

The binding of 3-methylcholanthrene (3-MC), a potent inducer of aryl hydrocarbon hydroxylase activity, to cytoplasmic proteins of a cloned rat hepatocyte culture, RL-PR-C, was studied by sucrose gradient centrifugation. Time course and dose-binding experiments performed on late-passage aryl hydrocarbon hydroxylase-inducible cultures indicate the presence of a saturable pool of high-affinity (average Kd, 3.6 nm) binding sites in the cytosol of these cells. The number of binding sites varied from 20,000 to 80,000 per late-passage hepatocyte with a total capacity of approximately 2.2 pmol of 3-MC bound per mg of cytosolic protein. The complex sedimented at 4.0 +/- 0.2S regardless of the ionic strength of the homogenization buffer or gradient solutions. It was sensitive to denaturation by sodium dodecyl sulfate and trypsin but not by DNase I, RNase A, or the nonionic detergent Nonidet P-40. The binding of 3-MC to the protein was inhibited by 1,2-benzanthracene, benzo(a)pyrene, 5,6-benzoflavone, and 7,8-benzoflavone but not by a series of steroids, aflatoxin B1, phenobarbital, or Aroclor 1254. Elevating the temperature of cultures cells to 37 degrees after the standard ligand-binding incubation at 4 degrees resulted in a rapid decrease in cytoplasmic saturable binding and a concomitant increase in nuclear- and chromatin-associated ligand. A portion of this nuclear-associated ligand was extractable with 400 mM KCl. Adsorption of the [3H]-3-MC binding complex by nuclei in vitro suggested that the 4S binding protein facilitated the entry of 3-MC into the nucleus. The presence of the 4S binding species correlated with the level of inducibility of aryl hydrocarbon hydroxylase throughout its development in RL-PR-C and therefore may be involved in the process of induction of this enzyme.

Morphological transformation and DNA adduct formation by benz[j]aceanthrylene and its metabolites in C3H10T1/2CL8 cells: evidence for both cyclopenta-ring and bay-region metabolic activation pathways.

Benz[j]aceanthrylene (B[j]A), a cyclopenta-fused polycyclic aromatic hydrocarbon related to 3-methylcholanthrene, has been studied to identify the major routes of metabolic activation in transformable C3H10T1/2CL8 (C3H10T1/2) mouse embryo fibroblasts in culture. Previous studies have reported that the major (55% of total) B[j]A metabolite formed by C3H10T1/2 cells was (+/-)-trans-9,10-dihydro-9,10-dihydroxy-B[j]A (B[j]A-9,10-diol), the dihydrodiol in the bay-region ring, with moderate amounts (14% of total) of (+/-)-trans-1,2-dihydro-1,2-dihydroxy-B[j]A (B[j]A-1,2-diol), the cyclopenta-ring dihydrodiol. The morphological transforming activities of three potential intermediates formed by metabolism of B[j]A by C3H10T1/2 cells, (+/-)-anti-trans-9,10-dihydro-9,10-dihydroxy-B[j]A-7,8-oxide (B[j]A-diol-epoxide), B[j]A-9,10-oxide, and B[j]A-1,2-oxide as well as the two B[j]A-dihydrodiols were examined. B[j]A, B[j]A-diol-epoxide, B[j]A-1,2-oxide, and B[j]A-9,10-diol were found to have moderate to strong activities with B[j]A-diol-epoxide the most active compared to B[j]A, while B[j]A-1,2-diol was inactive. B[j]A-9,10-oxide was found to be a weak transforming agent. At 0.5 microgram/ml, the following percentage of dishes with type II or III foci were observed: B[j]A, 59%; B[j]A-diol-epoxide, 75%; B[j]A-1,2-oxide, 25%; and B[j]A-9,10-diol, 17%. DNA adducts of B[j]A, B[j]A-9,10-diol, B[j]A-diol-epoxide, B[j]A-9,10-oxide, and B[j]A-1,2-oxide in C3H10T1/2 cells were isolated, separated, identified, and quantitated using the 32P-postlabeling method. B[j]A forms two major groups of adducts: one group of adducts is the result of the interaction of B[j]A-1,2-oxide with 2'-deoxyguanosine and 2'-deoxyadenosine; the second group of adducts is a result of the interaction of B[j]A-diol-epoxide with 2'-deoxyguanosine and 2'-deoxyadenosine. Qualitative and quantitative analysis of the postlabeling data suggests that B[j]A is metabolically activated by two distinct routes, the bay-region diol-epoxide route and the cyclopenta-ring oxide route, the former being the most significant.

Carcinogen-binding proteins in the rat ventral prostate: specific and nonspecific high-affinity binding sites for benzo(a)pyrene, 3-methylcholanthrene, and 2,3,7,8-tetrachlorodibenzo-p-dioxin.

The polychlorinated dibenzodioxin [3H]-2,3,7, 8-tetrachlorodibenzo-p-dioxin (TCDD) and the carcinogens [3H]benzo(a)pyrene and [3H]-3-methylcholanthrene bound to saturable binding sites in cytosol from the rat ventral prostate. Analysis of equilibrium binding parameters in diluted cytosol preparations indicated an apparent Kd of approximately 2 nM and a binding capacity of approximately 1 nmol/mg cytosolic protein, corresponding to approximately 5% of the total protein content. However, gel permeation chromatography analysis as well as velocity sedimentation analysis on sucrose gradients of [3H]TCDD-labeled rat prostatic cytosol indicated binding of [3H]TCDD to two discrete species. These analyses indicated a sedimentation coefficient of 3.6-3.8S, a Stokes radius of 25-28 A, and a calculated relative molecular weight of 42,000-45,000 for the most abundant binding species. The other binding species sedimented at 4-5S under high ionic strength conditions and at 8-10S under low ionic strength conditions and had a Stokes radius of approximately 60 A, a relative molecular weight of approximately 100,000 and an estimated concentration of 5-20 fmol/mg cytosolic protein. Binding of [3H]TCDD to this species was displaceable by a 200-fold M excess of 2,3,7,8-tetrachlorodibenzofuran. Therefore, this species was tentatively identified as the TCDD receptor. The properties of the high-capacity binder of [3H]TCDD were found to be similar to the characteristics of a protein previously purified from the rat ventral prostate, prostatic secretory protein, which binds androgens as well as estramustine, a nitrogen mustard derivative of estradiol. The binding of estramustine to diluted prostatic cytosol was shown to be competitively inhibited by 2,3,7,8-tetrachlorodibenzofuran. Moreover, purified prostatic secretory protein bound [3H]TCDD, [3H]benzo(a)pyrene, as well as [3H]-3-methylcholanthrene. It is suggested that binding to this protein is responsible for the high-binding capacity of carcinogens in cytosol from the rat ventral prostate.

Modification of transplacental tumorigenesis by 3-methylcholanthrene in mice by genotype at the Ah locus and pretreatment with beta-naphthoflavone.

Transplacental lung and liver tumorigenesis in the mouse by 3-methylcholanthrene (MC) was assessed as a function of inducibility of MC metabolism in fetus and in mother, and of pretreatment of the mothers with a noncarcinogenic inducer, beta-naphthoflavone (beta NF). Pregnant (C57BL/6 X DBA/2)F1 females (genotype Ahb Ahd, inducer responsive) mated to DBA/2 males received 45 or 100 mg/kg MC on gestation day 17, and DBA/2 females (genotype Ahd Ahd, nonresponsive) mated to F1 males were given 5 or 30 mg MC/kg. These crosses generated both responsive and nonresponsive offspring. Phenotype and tumor incidences were determined at 13 months of age. The transplacental action of MC was dose dependent and resulted in more lung and liver tumors in induction-responsive offspring than in nonresponsive littermates in most comparisons. beta NF alone did not result in increased numbers of tumors. Significant, complex effects were seen when the mothers were pretreated with beta NF (150 mg/kg) on gestation day 15, before MC on day 17. The beta NF pretreatment protected the fetuses of the F1 mothers: there was a significant overall 30 to 50% reduction in numbers of lung and liver tumors. The greatest effect was seen in the induction-responsive males, who experienced a 50% reduction in both incidence and multiplicity of lung tumors after 100 mg MC/kg, compared with males exposed to MC only. By contrast, beta NF pretreatment of DBA mothers had no general effect but rather potentiated the action of the 5 mg MC/kg dose on multiplicity of lung tumors in inducible males, causing a significant 4-fold increase. It also caused a 60% increase in inducible male liver tumor multiplicity when given before the 30 mg MC/kg dose. Thus, beta NF pretreatment was protective when the mother was inducible, especially in the inducible fetuses of such a mother, but when the mother was noninducible the beta NF pretreatment had no effect in some situations and potentiated the action of the carcinogen in others, mainly in inducible fetuses. These results underscore the fact that induced maternal and fetal metabolism contribute to risk of transplacental tumorigenesis by MC in qualitatively opposite ways.

Uptake, metabolism, and persistence of 3-methylcholanthrene in rat embryo cells infected with murine leukemia virus.

The uptake and persistence of 3-methylcholanthrene have been followed in both unifected rat embryo tissue culture cells and in cells infected with type C RNA virus. No significant differences in these parameters were observed as a function of viral infection or cell passage level. Moreover, neither binding of 3-methylcholanthrene to nucleic acids or proteins nor carcinogen metabolism were altered by the viral carrier state. Although transformation of rat cells by chemical carcinogens alone has been reported by us and other authors, the low-passage rat embryo cells used in this study will not transform unless cells are carrying exogenous type C RNA virus. We thus suggest that the virus must play a more direct role in the transformation process rather than affecting the ability of the cell to absorb, retain, or metabolize the chemical.

Polycyclic aromatic hydrocarbon toxicity and induction of metabolism in cultivated esophageal and epidermal keratinocytes.

Serially cultivated keratinocytes of human and rat epidermis and esophagus were compared with respect to their sensitivity to toxic effects of 3-methylcholanthrene and ability to metabolize benzo(a)pyrene. 3-Methylcholanthrene was highly toxic to the human keratinocytes and to early-passage rat epidermal keratinocytes, as evidenced by markedly reduced growth upon continuous exposure or reduced colony-forming ability after 1-day exposure to concentrations of 0.4 to 40 microM in the culture medium. Rat esophageal and late-passage rat epidermal cells appeared insensitive to 3-methylcholanthrene by these criteria. All the cell types except late-passage rat epidermal cells metabolized benzo(a)pyrene at comparable rates, and expressed aryl hydrocarbon hydroxylase maximally inducible by similar concentrations of 3-methylcholanthrene. Biotransformation in the human cells was greatly inhibited by alpha-naphthoflavone, a specific inhibitor of aryl hydrocarbon hydroxylase. The lack of toxicity of 3-methylcholanthrene toward late-passage rat epidermal cells can be attributed to the low constitutive rate of biotransformation these cells exhibit. The insensitivity of rat esophageal cells despite substantial metabolic activity reflects the importance of intrinsic differences among keratinocytes derived from different epithelia and species in determining toxic response. Human cervical and monkey esophageal keratinocyte cultures also actively metabolized benzo(a)pyrene, illustrating further the utility of the culture system for exploring differences among species and epithelial cell types.

Effect of diet on fecal excretion and gastrointestinal tract distribution of unmetabolized benzo(a)pyrene and 3- methylcholanthrene when these compounds are administered orally to hamsters.

3-Methylcholanthrene (3MC) administered p.o. has induced tumors of the hamster gastrointestinal tract (GIT), including the large intestine. This process may depend on the concentration of unchanged hydrocarbon in the GIT contents. Benzo(a)pyrene (BP) ingestion could be involved in human GIT carcinogenesis. Accordingly, male Syrian golden hamsters were fed diets containing BP or 3MC for 10 days. Feces collected during the last two to three days of feeding were analyzed for the unchanged hydrocarbons by KOH:methanol digestion, Florisil column and paper chromatography, and ultraviolet spectrophotometry. With a semisynthetic diet containing 5% Alphacel, 6% corn oil, and 100 microgram BP per g. fecal BP excretion was 0.45% of the dose. Variation of the corn oil content had little effect. Fecal BP excretion was increased 13 times (to 6% of the dose) when 5% wheat brain was used in place of Alphacel and 4.5 times when a commercial diet was used. This suggests that bran adsorbed or sequestered the BP. Water content of the large-intestine contents was increased when the brain diet was fed. Both these factors could affect mucosal exposure to BP. For 3MC, fecal excretion of unchanged hydrocarbon was 14 times greater than for BP under similar conditions. The GIT contents of hamsters fed BP or 3MC showed hydrocarbon concentrations in the order: stomach greater than lower large intestine greater than other sections.

Age-, tissue-, and Ah genotype-dependent differences in the binding of 3-methylcholanthrene and its metabolite(s) to mouse DNA.

The induction of transplacental carcinogenesis by 3-methylcholanthrene (MC) in mice is determined, in part, by the genotype at the Ah locus. The relationship of Ah genotype and MC-induced DNA adducts was tested by comparing the response of pregnant and fetal C57BL/6 mice (Ahb Ahb; responsive to the induction of MC metabolism) and DBA/2mice (Ahd Ahd; nonresponsive). On day 17 of gestation (day 1 = presence of vaginal plug), C57BL/6 mice were treated i.p. with 100 mg/kg MC and DBA/2 mice with 30 mg/kg. Mice were sacrificed 24 h later and the tissues were analyzed for the presence of DNA adducts using the P1 nuclease version of the 32P-postlabeling method. With a 3.3-fold difference in administered dose, the total adduct levels in fetal DNA were (a) similar in both strains with the exception of liver, for which C57BL/6 mice had more adducts; (b) higher in the lung than skin, liver, or thymus; and (c) only 1/4 to 1/14 of the adult levels. Maternal DBA/2DNA contained more adducts in the thoracic lymph nodes and liver but fewer in the placenta and lung, compared to maternal C57BL/6 DNA. More adducts were detected in lung DNA than liver DNA in C57BL/6 mice. In contrast, these levels were similar in DBA/2 mice. When the difference in dose administered was considered in conjunction with this, less MC bound to DNA of C57BL/6 than DBA/2 mice overall. To identify adducts, oxidized metabolites of MC, 1-hydroxy-, 2-hydroxy-, 9,10-dihydrodiol-, or 3-methoxymethyl-MC, were topically applied to the dorsal skin of both strains. All of these metabolites produced adducts. Approximately 14 different adduct spots were detected. The two most abundant adducts were produced by 1-hydroxy-, 2-hydroxy-, and 9,10-dihydrodiol-MC. One of these also contained a 3-hydroxymethyl group. Several adducts did not contain the 9,10-dihydroxy group. The adducts derived from 3-methoxymethyl-MC were consistently found in greater abundance in DNA from C57BL/6 tissues, compared with DBA/2. Thus, oxidation of the 3-methyl group may be enhanced by Ah-dependent induction of MC metabolism. Together, these results suggest that the individual and total adduct levels are influenced by the genotype at the Ah locus, the route of administration, and the metabolite(s) with tissue and age specificity.

Effects of metabolism on the binding of polycyclic hydrocarbons to nuclear subfractions of cultured AKR mouse embryo cells.

A high-affinity localization of [2H]methylcholanthrene and/or its metabolites to a specific nuclear fraction (Fraction l) between 4 and 72 hr of exposure is described. Other carcinogenic hydrocarbons, such as benzo(a)pyrene and dibenz(a,h)anthracene, demonstrate a similar markded localization in Fraction l after 24 hr of incubation. The weak carcinogen dibenz(a,c)anthracene, as well as steroid hormones, sho little localization in this fraction. Two types of binding are measured:and organic solvent: extractable (non-covalent) binding and a nonextractable (covalent) binding. Maximal levels of the combined extractable and nonextractable binding per mass DNA are found at 24 hr of exposure, while at 48 and 72 hr of exposure the binding is reduced. The highest level of the nonextractable binding per mass DNA is also observed at the 24-hr exposure period. However, as the period of exposure increases, the proportion of the total nuclear-bound radioactivity representing the non-extractable type increases. Analysis by high-pressure liquid chromatography of the extractable radioactivity from the fractions indicates that the longer the period of exposure, the greater the extent of metabolism of 3-methylchol-anthrene. When 7,8-benzoflavone (a flavanoid hydroxylase inhibitor) is included in the incubations, practically all metabolic alterations of the parent compound are prevented. In addition, the time-dependent increase in nonextractable radioactivity from all nuclear subfractions is prevented. A metabolic-dependent "covalent" binding of carcinogenic polycyclic aromatic hydrocarbons to the barious nuclear subfractions of chromatin is suggested. This covalent binding is markedly localized in a specific fraction of the chromatin containing rapidly labeled nascent RNA.