Role of the maternal environment in determining susceptibility to transplacentally induced chemical carcinogenesis in mouse fetuses.

Treatment of pregnant mice with 3-methylcholanthrene (MC) causes lung and liver tumors in the offspring, the incidences of which are greatly influenced by the Ah locus regulated induction phenotype for aryl hydrocarbon hydroxylase activity (AHH) in both the mother and fetuses. In order to examine the biochemical and molecular mechanisms responsible for the modulating effect of maternal environment on tumor susceptibility, reciprocal crosses between responsive C57BL/6 and non-responsive DBA/2 mice were made and the pregnant mothers were treated i.p. on the 17th day of gestation with either olive oil alone, 30 mg/kg of MC, or 30 mg/kg of beta-naphthoflavone (beta NF). At various times after injection, the mothers were killed and the fetuses removed for enzymatic and molecular blot analysis. In fetal lung tissues, the absolute levels and relative induction ratios of AHH activity from D2B6F1 fetuses were very similar to those obtained in B6D2F1 fetuses during the first 24 h following a transplacental exposure to either inducing agent. This was also the case 48 h after an injection of beta NF. However, 48 h after exposure to MC, the AHH activity in fetal lungs from B6 mothers had declined to practically control values, whereas fetal lungs from D2 mothers still exhibited a high level of AHH activity. Similar induction kinetics for the CYPIA1 gene were obtained in fetal livers. These results were confirmed at the RNA level by quantitative slot-blot analysis of fetal RNA preparations. In both organs, treatment with inducing agents for the P450IA1 gene resulted in a rapid and early induction of CYPIA1 RNA by 4 h. Fetuses from D2 mothers, however, showed a more sustained induction of CYPIA1 RNA following exposure to MC than did fetuses from B6 mothers. These results suggest that the observed increase in tumor susceptibility observed in the offspring of D2 mothers compared to the offspring of B6 mothers was due, at least in part, to the differences in the persistence of induction of the CYPIA1 gene locus, and may be the result of differences in the clearance rates of MC from the fetal and maternal compartments or its pharmacokinetic distribution in the two types of maternal environments.     

Differential induction of fetal mouse liver and lung cytochromes P-450 by beta-naphthoflavone and 3-methylcholanthrene

Previous studies have shown that the incidences of liver and lung tumors in mice exposed transplacentally to 3-methyl-cholanthrene (MC) were significantly influenced by the sensitivity of both mothers and fetuses to induction of cytochrome(s) P-450 by polycyclic aromatic hydrocarbons. In order to delineate further the biochemical and molecular processes underlying the observed biological effects, the inductive effect of MC and beta-naphthoflavone (beta NF) on cytochrome P-450 was determined at the biochemical and molecular levels. C57BL/6 females were mated with DBA/2 males and treated i.p. on day 17 of gestation with olive oil alone, 150 mg/kg of beta NF or different doses of MC. At various times after injection the mothers were sacrificed and the fetuses removed for biochemical and molecular studies. MC caused maximal induction of aryl hydrocarbon hydroxylase (AHH) activity by 8 h in both the liver and lung. beta NF caused nearly maximal induction of AHH activity by 8 h in the lung but had little effect on liver AHH activity at this time. Maximal induction with beta NF occurred by 24 h in both organs. Addition of monoclonal antibody 1-7-1, specific for the MC-inducible forms of cytochrome P-450 (P-450IA1 and A2), to the incubation mixtures resulted in a 55-70% inhibition of AHH activity in both lung and liver assays, regardless of the inducing agent used, while having no effect on AHH activity from oil-treated mice. RNA blot analysis carried out in parallel with enzyme assays demonstrated that the levels of enzyme activity correlated very well with the levels of steady-state RNAs. MC caused maximal induction of P-450IA1 RNA levels 4 h after injection in both organs and a biphasic secondary increase was observed in the lung. Maximal levels of P-450IA1 RNA were seen at 12-16 h following injection of beta NF. However, the ratio of P-450IA1 RNAs present at 16 versus 2 h in the beta NF-treated liver appeared greater than that in the lung. P-450IA2 was also induced in fetal liver and lung, but at low levels relative to P-450IA1. The results indicate that the increase in functional AHH activity was primarily due to induction of cytochrome P-450IA1. The differences in induction kinetics observed for cytochromes P-450IA1 and A2 suggest that these enzymes exhibit both tissue- and inducer-dependent specificity.     

CARCINOGENESIS: Mouse lung tumors exhibit specific Ki-ras mutations following transplacental exposure to 3-methylcholanthrene

A pharmacogenetic mouse model was utilized to determine therole of Cyplal expression on the formation of Ki-ras mutationsin lung tumors following transplacental exposure to polycyclicaromatic hydrocarbons (PAHs). A backcross between Ah responsivemale B6D2F1 mice and non-responsive female DBA mice resultedin a litter in which both responsive and nonresponsive fetusesresided in the same nonresponsive maternal environment Pregnantmothers received a single i.p. injection of either 10 or 30mg/kg of 3-methylcholanthrene (MC) or olive oil vehicle on day17 of gestation. At the higher dose of MC, the responsive offspringof both sexes had significantly (P < 0.05) higher incidencesof lung tumors than their nonresponsive littermates. The maleresponsive mice also exhibited a significantly increased livertumor incidence over the nonresponsive mice at the P / 0.05level. Administration of 10 mg/kg of MC caused a very low incidenceof lung tumors and did not result in the appearance of macroscopicallyvisible liver tumors. Exons 1 and 2 of the Ki-ras gene wereamplified from paraffin-embedded tissue samples. The PCR productswere screened by allele-specific oligonucleotide hybridization(ASO). Thirteen of 16 lung tumors (81%) screened exhibited pointmutations in the 12th or 13th codon, including seven tumorsthat contained GGT     

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.     

Metabolism of transplacental carcinogens

Literature on fetal metabolism of carcinogens since 1980 has been systematically listed and selectively discussed. The published data continues to support the conclusion that animal and human fetal tissues have the capacity to metabolize carcinogens, but at a low rate compared to the adult. Metabolism of low-molecular-weight chemicals, including nitrosamines, appears only near term in the rodent and is poorly inducible transplacentally; these agents are correspondingly relatively ineffective as fetal carcinogens. Metabolism of aromatic carcinogens, by contrast, appears early in gestation and is highly inducible transplacentally in rodents by chemicals such as polycyclic aromatic hydrocarbons (PAH) and tetrachlorodibenzo-p-dioxin, resulting in dramatic percent increases in enzyme activity. Transplacental induction has not been unequivocally demonstrated for the human fetus. Phase II enzymes, metabolizing aromatic compounds to water-soluble forms, generally have higher constitutive activity but lower degree of inducibility in the fetus, compared with phase I (activating) enzymes, and appear to show quantitatively different patterns in the human compared with the rodent. Specific and sensitive new technologies, including 32P-postlabelling, immunodetection of specific proteins, and use of cDNA probes, are beginning to be applied to fetal systems and are providing a more definitive and detailed understanding of the ontogeny and modulation of fetal carcinogen metabolizing enzymes. Fetal and maternal metabolism of PAH, especially methylcholanthrene (MC), have been found to be important determinants in susceptibility of the fetus to tumorigenesis. In particular, we have utilized a mouse model system wherein a single dominant gene, Ah, confers responsiveness to induction of PAH metabolism by cytochrome Pl450 (IA1); the recessive allele, Ah, is associated with nonresponsiveness. In appropriate backcrosses between C57BL/6 (AhAh) and DBA/2 (AhAh) mice, responsive and nonresponsive fetuses were carried together in mothers who were, themselves, either responsive or nonresponsive. In both cases, responsive fetuses later developed more lung and liver tumours after transplacental MC, compared with nonresponsive littermates. Fetuses of responsive mothers, however, experienced a lower cancer risk than did those of nonresponsive mothers at a comparable MC dose. Pretreatment of the pregnant mice with the noncarcinogenic inducer, beta-naphthoflavone (BNF) had a uniform protective effect for all of the fetuses, especially the responsive ones, if the mother was responsive. For nonresponsive mothers, by contrast, BNF pretreatment led to an enhancement of tumorigenesis, in the responsive fetuses only, under certain conditions of dose and fetal sex.(ABSTRACT TRUNCATED AT 400 WORDS)     

Effect of pretreatment with beta-naphthoflavone on tumorigenesis by N-nitrosoethylurea in five mouse strains

The non-carcinogenic inducer of the Ah locus, beta-naphthoflavone (beta NF), was administered to females of 5 mouse strains at a dose of 150 mg/kg 48 h before treatment with a tumorigenic dose of the direct-acting carcinogen, N-nitrosoethylurea (ENU), once weekly for 4 weeks. The strains used were C57BL/6, C3H/He and NIH Swiss (responsive to Ah locus induction) and AKR and DBA/2 (induction-non-responsive). The ENU caused primary lung tumors in all strains and in some cases smaller numbers of other neoplasms, including lymphomas, sarcomas and hepatocellular tumors. The beta NF pretreatment did not reduce the numbers of any of the tumors, compared with mice given ENU alone. This result is in contrast to previous findings of a strong protective effect of beta NF against tumorigenesis in Ah-responsive strains by the metabolism-dependent carcinogens, benzo[a]pyrene and 3-methylcholanthrene and confirms that this protection is directly related to enzyme induction. beta NF treatment caused a significant doubling in the number of lung tumor bearers among the ENU-exposed C57BL/6 mice but in no other strain, suggesting the possibility of strain-specific tumor promotion.     

Fetal mouse susceptibility to transplacental lung and liver carcinogenesis by 3-methylcholanthrene: positive correlation with responsiveness to inducers of aromatic hydrocarbon metabolism

The role of metabolic activation of carcinogens in fetal tissueas a determinant of sensitivity in transplacental carcinogenesiswas investigated in a pharmacogenetic experiment utilizing backcrossesof C57BL/6 (Ah^bAh^b, responsive to induction of aromatic hydrocarbonmetabolism) and DBA/2 (Ah^dAh^d, non-responsive) mice. Responsive(C57BL/6 x DBA/2)F₁ and non-responsive DBA mothers, all carryingboth responsive (Ah^bAh^d) and non-responsive (Ah^dAh^d) fetuses,were given i.p. doses of the carcinogen 3-methylcholanthrene(MC) ranging from 5 to 175 mg/kg on gestation day 17. At 10months of age the metabolic phenotype of each offspring wasdetermined, and correlated with number of lung and liver tumors.Both male and female Ah^bAh^d (responsive) offspring in most dosegroups presented a consistent two- to three-fold higher incidenceof lung tumors than did non-responsive Ah^dAh^d littermates. Thedifference held for offspring of both (C57BL/6 x DBA)F₁ andDBA mothers and it was of statistical significance for one orboth sexes at most dosage levels. Hepatocellular tumors werealso significantly more frequent in responsive male Ah^bAh^d progenyof (C57BL/6 x DBA/2)F₁ mothers than in non-responsive Ah^dAh^dlittermates. Progeny of the DBA mothers exhibited significantlymore liver and lung tumors than did those of the (C57BL/6 xDBA/2)F₁ mothers receiving the same dose. These results suggestthat in this model system both maternal and fetal genotype forresponsiveness to induction of aromatic hydrocarbon metabolismare important factors modulating fetal carcinogenic risk.     

Strain-dependent lung tumor formation in mice transplacentally exposed to 3-methylcholanthrene and post-natally exposed to butylated hydroxytoluene.

The carcinogenic effects of in utero exposure to 3-methylcholanthrene (MC) have been demonstrated in the tumor-resistant C57BL/6 (B6) and DBA (D2) strains of mice. In this study, we determined the effects of in utero exposure to MC in BALB/c mice, a strain which demonstrates greater susceptibility to lung tumor induction, and compared our findings with those previously found in [D2xB6D2F(1)]F(2) mice. In addition, we assessed the molecular pathogenesis of the chemically induced tumors and examined the effects of the putative lung tumor promoter butylated hydroxytoluene (BHT) in BALB/c mice. BALB/c mice were treated on day 17 of gestation with 5, 15 or 45 mg/kg MC and 6 weeks after birth with BHT for 6 consecutive weeks. Mice were killed at 6 months of age. Ki-ras, p16Ink4a and p19ARF gene loci were amplified from paraffin-embedded lung tumor tissue and screened for the presence of point mutations via allele-specific oligonucleotide hybridization and single strand conformation polymorphism (SSCP) analyses. Ki-ras point mutations were found in 56% (20/36) of BALB/c lung tumors, with 33% (2/6) of the hyperplasias, 58% (10/19) of the adenomas and 73% (8/11) of the carcinomas exhibiting point mutations at this gene locus. Similar incidences of Ki-ras mutations were previously found following transplacental exposure of [D2xB6D2F(1)]F(2) mice to MC and treatment of adult A/J mice with urethane. Interestingly, a strain-dependent difference was observed in the mutational spectrum. Sixty-two and 38% of the lung lesions in BALB/c mice exhibited G-->C and G-->T transversions, respectively, in contrast to the 13 and 84% incidences previously observed in [D2xB6D2F(1)]F(2) mice. SSCP analysis of the tumor suppressor gene p16Ink4a showed a 6% incidence of point mutations, consistent with that found in [D2xB6D2F(1)]F(2) mice. No mutations were found in exon 1beta of the p19ARF gene of either strain. BHT, a lung tumor promoter in adult mice, had no statistically significant effects on either tumor incidence, tumor multiplicity or the mutational spectrum produced in the Ki-ras gene by in utero MC treatment. However, though not significant, there was an observable trend in increased tumor multiplicity in mice co-treated with BHT. These data demonstrate the transplacental carcinogenic effect of MC in BALB/c mice and show that mutagenic damage to Ki-ras is a critical early event mediating murine lung tumorigenesis in both the tumor-sensitive and tumor-resistant strains. Unlike what occurs when adult BALB/c mice are treated with MC, BHT does not appear to significantly promote the formation of lung tumors following transplacental exposure to MC, possibly due to the rapid growth and cell proliferation in the developing organism. Strain-dependent differences in the Ki-ras mutational spectrum may be associated with their differential susceptibility to lung tumor initiation.     

Strain-specific induction of murine lung tumors following in utero exposure to 3-methylcholanthrene

Fetal mice are more sensitive to chemical carcinogens than are adults. We previously demonstrated that resistant offspring of a DBA/2 x (C57BL/6 x DBA2) backcross exhibited a high incidence of lung tumors 12-13 mo after transplacental exposure to 3-methylcholanthrene (MC). We compared the effects of in utero treatment with MC on lung tumor incidence in the offspring of intermediately susceptible BALB/c (C), resistant C57BL/6 (B6), and reciprocal crosses between these strains. Pregnant mice were treated with 45 mg/kg of MC on day 17 of gestation and tumor incidence, multiplicity, and the Ki-ras mutational spectrum determined in the offspring 12-18 mo after birth. Tumor incidences in C mice and reciprocal crosses were 86% and 100%, respectively, while B6 mice demonstrated resistance to tumorigenesis, with a tumor incidence of 11%. Tumor multiplicities in C, B6C, CB6, and B6 mice were 3.3 +/- 3.2, 5.8 +/- 3.2, 5.0 +/- 2.7, and <0.1, respectively. Ki-ras mutations, which occurred chiefly in the K(s) allele (96%), were found in 79-81% of reciprocally crossed F1 mice, 64% of C mice, and 50% of B6 mice, with the Val(12), Asp(12), and Arg(13) mutations associated with more aggressive tumors. A subset of these mice was used to demonstrate the utility of computer tomography (CT) for the visualization and measurement of lung tumors in the submillimeter range in vivo. Based on known genetic differences in murine strains for lung cancer, our results suggest the presence of a previously unidentified genetic factor(s) which appears to specifically influence lung tumorigenesis following exposure to carcinogens during fetal development.     

CD49f-based selection of circulating tumor cells (CTCs) improves detection across breast cancer subtypes

Dibenzo[def,p]chrysene (DBC) is a transplacental carcinogen in mice (15 mg/kg; gestation day (GD) 17). To mimic residual exposure throughout pregnancy, dams received four smaller doses of DBC (3.75 mg/kg) on GD 5, 9, 13 and 17. This regimen alleviated the previously established carcinogenic responses in the thymus, lung, and liver. However, there was a marked increase in ovarian tumors (females) and hyperplastic testes (males). [¹⁴C]-DBC (GD 17) dosing revealed transplacental distribution to fetal tissues at 10-fold lower concentrations than in paired maternal tissue and residual [¹⁴C] 3 weeks post-dose. This study highlights the importance of developmental stage in susceptibility to environmental carcinogens.     

In utero exposure of mice to dibenzo[a,l]pyrene produces lymphoma in the offspring: role of the aryl hydrocarbon receptor

Lymphoma and leukemia are the most common cancers in children and young adults; in utero carcinogen exposure may contribute to the etiology of these cancers. A polycyclic aromatic hydrocarbon (PAH), dibenzo[a,l]pyrene (DBP), was given to pregnant mice (15 mg/kg body weight, gavage) on gestation day 17. Significant mortalities in offspring, beginning at 12 weeks of age, were observed due to an aggressive T-cell lymphoblastic lymphoma. Lymphocytes invaded numerous tissues. All mice surviving 10 months, exposed in utero to DBP, exhibited lung tumors; some mice also had liver tumors. To assess the role of the aryl hydrocarbon receptor (AHR) in DBP transplacental cancer, B6129SF1/J (AHR(b-1/d), responsive) mice were crossed with strain 129S1/SvIm (AHR(d/d), nonresponsive) to determine the effect of maternal and fetal AHR status on carcinogenesis. Offspring born to nonresponsive mothers had greater susceptibility to lymphoma, irrespective of offspring phenotype. However, when the mother was responsive, an AHR-responsive phenotype in offspring increased mortality by 2-fold. In DBP-induced lymphomas, no evidence was found for TP53, beta-catenin, or Ki-ras mutations but lung adenomas of mice surviving to 10 months of age had mutations in Ki-ras codons 12 and 13. Lung adenomas exhibited a 50% decrease and a 35-fold increase in expression of Rb and p19/ARF mRNA, respectively. This is the first demonstration that transplacental exposure to an environmental PAH can induce a highly aggressive lymphoma in mice and raises the possibility that PAH exposures to pregnant women could contribute to similar cancers in children and young adults.     

Immunohistochemical determination of inducibility phenotype with a monoclonal antibody to a methylcholanthrene-inducible isozyme of cytochrome P-450

A monoclonal antibody (MAb) to a methylcholanthrene (MC)-induced cytochrome P-450, designated MAb 1-7-1, was used for immunohistochemical staining of formalin-fixed tissues from oil- and MC-treated C57BL/6, DBA/2, and [(C57BL/6 X DBA/2) F1 X DBA/2] F2 mice. An avidin-biotin-peroxidase complex immunohistochemical technique was used. For controls, the tissues were also exposed to MAbs 1-48-5 and HyHel-9 (to egg white lysozyme). In liver, MAb 1-7-1 specifically stained the cytoplasm of centrilobular hepatocytes of C57BL/6 mice treated with MC (80 mg/kg) 48 h before kill; staining was not observed with vehicle-treated C57BL/6 mice, with oil- or MC-treated DBA/2 mice, or with comparable antibody concentrations of control MAbs 1-48-5 or HyHel-9. In the F2 mice, about 50% were expected to be MC inducible (AhbAhd). Inducibility phenotype was determined by measuring the conversion of [14C]MC to oxidized and conjugated products by liver homogenates. In freshly fixed material from MC-treated mice, those livers shown by the determination of phenotype to be inducible also stained with MAb 1-7-1, whereas those not induced were immunohistochemically negative. Furthermore, there was a significant positive correlation between degree of staining and the level of MC-metabolizing activity measured biochemically. The immunohistochemical procedure was also accurate in determination of inducibility phenotype of livers that had been in paraffin blocks for up to 2 yr if more concentrated antibody was used. In lung, MAb 1-7-1 stained specifically the alveolar walls and endothelium of blood vessels in MC-induced C57BL/6 mice only; the control MAbs and other mice gave negative results. Similarly, in kidney MAb 1-7-1 stained only glomeruli and interstitial tissue of MC-induced C57BL/6 mice and only endothelium of blood vessels in the colons of these mice. These observations are consistent with induction of the cytochrome P-450 recognized by MAb 1-7-1 in the endothelial cells of extrahepatic tissue. Immunohistochemical staining with MAb thus shows great promise for highly specific localization of particular species of cytochromes P-450 in tissues, for in situ quantification of these enzymes, and for determination of inducibility phenotype with fixed material.     

Immunological identification and effects of 3-methylcholanthrene and phenobarbital on rat pulmonary cytochrome P-450

Rabbit antibodies to the phenobarbital (PB) inducible rat liver microsomal cytochrome P-450s b and e and to 3-methylcholanthrene (MC) inducible P-450c were used to examine the expression of these isozymes in rat lungs. Western blots of total lung microsomes demonstrated that about 40 pmol P-450b/mg protein (and no detectable P-450e) were present in lungs from control or MC treated rats and that pretreatment with PB caused a small but significant (P less than 0.05) increase in the expression of P-450b. Microsomes from control and PB treated lung contained minimal levels of P-450c, and MC induced this isozyme to 185 pmol/mg. Immunocytochemistry was used to demonstrate immunoreactivity to these isozymes in specific cell types. Neither P-450b nor P-450c was detectable in endothelial cells from control or PB treated lungs, but MC increased immunoreactivity to P-450c in pulmonary endothelial cells. Type II alveolar cells showed distinct immunoreactivity to P-450b and weak immunoreactivity to P-450c in control or PB treated rats. Individual Clara cells stained for either P-450c or P-450b in control, MC treated, and PB treated rats, and colocalization was observed in some cells. An increase in type II alveolar cell and Clara cell immunoreactivity to P-450c was observed after MC induction. Mast cells, identified by metachromatic Giemsa staining, appeared to react nonspecifically with both antisera. In conclusion, P-450c is highly inducible by MC in rat lung (detected in microsomes by Western blot), specifically in endothelial cells, Clara cells, and alveolar type II cells (as visualized by immunocytochemistry); and P-450b is present in rat lung microsomes, and immunoreactivity to this isozyme is localized in alveolar type II and Clara cells.     

Aryl hydrocarbon hydroxylase activity in mouse, rat, and human mammary tumors

Aryl hydrocarbon hydroxylase (AHH) activity was measured in microsomes from chemically induced and spontaneous mammary tumors of mice and rats and in 213 human breast tumors. Basal enzyme activities [pmol 3-hydroxybenzo(a)pyrene per mg protein per min] ranged from 0.05 to 0.5 for rat, 0.05 to 10 for mouse, and 0 to 40 for human tumors. For comparison, mean liver AHH activities were 13 in untreated rats and 100 in untreated mice. Thus, some human breast tumors had AHH activity exceeding that in rat liver. Injection of 80 mg beta-naphthoflavone per kg into tumor-bearing C3H/HeJ mice or Sprague-Dawley rats increased AHH activity to 10- to 70-fold over basal levels; there was no significant AHH induction in tumors from genetically "nonresponsive" DBA/2J or RF/J mice treated with beta-naphthoflavone, alpha-Naphthoflavone in the incubation flask inhibited AHH activity in some human breast tumors and stimulated activity in others, probably reflecting the presence of multiple forms of cytochrome(s) P-450 in the human tumor population. AHH activity in human tumors was not correlated with their estrogen receptor content. Since several drugs used in cancer treatment are substrates for polysubstrate monoxygenases, high levels of AHH activity in some human tumors may play a role in their response to chemotherapy.     

Reaction of 7,12-dimethylbenz(a)anthracene with DNA of fetal and maternal rat tissues in vivo.

Pregnant BD-IX rats (21st day of gestation) received a single IV injection (15 mg/kg) of tritiated 7,12-dimethylbenz(a)anthracene (DMBA), A DOSE KNOWN TO INduce a high incidence of nervous-system tumors in the offspring. The animals were killed 12 h later and hydrocarbon-deoxyribonucleoside products from DNA of maternal and fetal tissues were separated on Sephadex LH-20 columns eluted with a 20-100% methanol gradient. Concentrations of the major DMBA-DNA adduct varied considerably, with highest values in maternal intestine, liverand lung, followed by spleen, kidney and brain. In fetal intestine and liver, concentrations were 34% and 16% lower than in the respective maternal organs whereas the reaction with cerebral DNA was 2 1/2 times higher in fetuses than in the pregnant mother. This indicates that there is no significant placental barrier to DMBA or DMBA metabolites involved in DNA binding and that rat fetuses participate in the metabolic formation of the ultimate carcinogen.     

Effects of polychlorinated biphenyls on lung and liver tumors initiated in suckling mice by N-nitrosodimethylamine

A mixture of polychlorinated biphenyls (PCB), Aroclor 1254, was administered ip (500 mg/kg) to pregnant Swiss noninbred CD-1 mice on the 19th day of gestation. The suckling offspring of these mice and of controls were then treated ip with 5 mg N-nitrosodimethylamine (DMN)/kg on postnatal day 4 or day 14. The progeny were killed at 28 weeks or 18 months of age. The DMN treatment caused lung and liver tumors. Among the mice given DMN on day 14, exposure also to PCB resulted in a lower tumor incidence in some of the treatment groups. This protective action was most evident for lung tumors in both sexes at 18 months of age and for liver tumors in males at 28 weeks. However, the PCB exposure also led to significant increases in the percentage of mice with extensive DMN-initiated liver tumors at 18 months of age compared to the results obtained when only DMN was given. This effect was most pronounced in mice given DMN on postnatal day 4.     

Strain-dependent differences in the metabolism of 3-methylcholanthrene by maternal, placental, and fetal tissues of C57BL/6J and DBA/2J mice

C57BL/6J (B6) and DBA/2J (D2) mice have different susceptibilities to developmental toxicity and transplacental carcinogenesis induced by in utero exposure to polycyclic aromatic hydrocarbons, which has been associated with polycyclic aromatic hydrocarbon metabolism and inducibility at the Ah locus. The distribution of total 3-methylcholanthrene (3-MC)-associated radioactivity in maternal, placental, and fetal tissues of beta-naphthoflavone-pretreated pregnant B6 and D2 mice was determined up to 12 h after p.o. exposure to [6-14C]-3-MC (63 mg/kg, 20 mu Ci) on gestational day 17. 3-MC-associated radioactivity in maternal plasma was not significantly different in the two strains. However, D2 tissue homogenates had consistently higher levels of 3-MC-associated radioactivity, which included both bound and free parent compound and metabolites. Increased metabolism of 3-MC by B6 maternal liver was suggested by the induced levels of aryl hydrocarbon hydroxylase activity in that tissue and by the observation that levels of total radioactivity decreased more rapidly in B6 tissues than in D2 tissues. The D2 fetal lung, the target tissue for 3-MC-induced transplacental carcinogenesis, appeared to accumulate 3-MC-associated radioactivity for a longer period of time than either the D2 fetal liver or the B6 fetal tissues. This study suggests that the genetic differences in fetal susceptibility to the developmental toxicity and transplacental carcinogenesis of 3-MC may be related to the presystemic elimination of the compound from both maternal and fetal tissues.     

The 4S polycyclic aromatic hydrocarbon-binding protein: immunohistochemical localization in mice

The 4S polycyclic aromatic hydrocarbon (PAH)-binding protein (PBP) is a cytoplasmic protein that binds PAHs with specificity and high affinity. We have used antisera for the PBP and unlabeled peroxidase anti-peroxidase immunohistochemistry to demonstrate its possible localization in cell types known to have xenobiotic metabolizing capabilities. Cellular sites of the PBP in liver, lung and kidney of C57BL/6 and DBA/2 mice were probed. The PBP was visualized in hepatocytes throughout the liver lobule and was not preferentially located in either centrilobular or periportal areas. However, cellular heterogeneity with respect to PBP content was clearly evident in the hepatocyte population. The positive reactivity correlated with substantial levels of benzo[a]pyrene (B[a]P) binding in liver cytosol. In the lung, the PBP was found in the bronchiolar epithelium and the alveolar septa, and was localized in ciliated and non-ciliated Clara and alveolar type II cells as well as in alveolar macrophages. In the kidney, the glomeruli and epithelia of proximal and distal convoluted tubules and collecting ducts were labeled. Staining for the PBP was greatest in the apical region of the pyramid and was localized in the epithelial lining of the collecting ducts. Relatively lower levels of the PBP were detected in the lung and kidney than in the liver. Staining was localized in the cytoplasmic compartment of cells in all tissues examined. Similar immunoreactivities were exhibited in the tissues of both C57BL/6 and DBA/2 mice. Treatment with beta-naphthoflavone (beta NF) altered neither the intensity nor pattern of immunostaining. Furthermore, treatment with beta NF or isosafrole has no effect on the Kd and Bmax of B[a]P binding to liver cytosolic PBP. The results of our experiments demonstrate localization of the PBP to sites of active physiological response to PAH exposure.     

Pancreatic islet-cell and other tumors in rats given heliotrine, a monoester pyrrolizidine alkaloid, and nicotinamide.

Three rats of six males, surviving 22 to 27.5 months after one or two intragastric doses of the monoester pyrrolizidine alkaloid, heliotrine (230 mg/kg body weight), and pretreatment with nicotinamide (350 mg/kg body weight) by pretreatment with nicotinamide (350 mg/kg body weight) by i.p. injections, developed pancreatic islet-cell tumors, accompanied in one of the rats by transitory cell papillomas of the urinary bladder and interstitial testicular tumors and in another by a hepatoma. The lesions in the livers showed progression from megalocytosis, to microscopic hepatocellular hyperplasia, to increasingly larger nodules and hepatoma. One rat, given heliotrine, but no nicotinamide, also developed adenoma of the pancreatic islet cells. Adenomas of the pituitary were present among the experimental and also among the control rats killed between 19 and 27.5 months after the beginning of the experiment, and they are not likely to have been caused by the alkaloid. Heliotrine, in which the crucial double bond in the pyrrolizidine moiety is sterically hindered, appears to be less readily sequestered by the liver and also to affect other organs. Alkylation of nicotinamide at the N-1 position prevents its being reused for coenzyme biosynthesis. Hence, pretreatment of rats with large doses of nicotinamide prevents the depletion of nicotinamide adenine dinucleotide coenzymes and liver necrosis in rats given heliotrine (230 mg/kg body weight).     

Induction of tumors of the liver, lung, ovary and adrenal in adult mice after brief maternal gestational exposure to inorganic arsenic: promotional effects of postnatal phorbol ester exposure on hepatic and pulmonary, but not dermal cancers

Arsenic is a recognized human carcinogen and development of rodent models remains a critically important research objective. Since gestation can be a period of high sensitivity to chemical carcinogenesis, we have performed a series of transplacental carcinogenicity studies in mice with inorganic arsenic. In this study, groups of pregnant C3H mice received drinking water containing sodium arsenite (NaAsO2) at 0, 42.5 and 85 p.p.m. arsenic ad libitum from days 8 to 18 of gestation. These doses of arsenic were well tolerated. Dams delivered normally and at weaning (4 weeks) offspring were randomly put into groups (n = 25) of males or females according to maternal dose. In an attempt to promote skin cancers initiated by transplacental arsenic, duplicate groups of control or arsenic exposed offspring were topically exposed to 12-O-tetradecanoyl phorbol-13-acetate (TPA; 2 micro g/0.1 ml acetone, twice/week) from 4 to 25 weeks of age. Irrespective of TPA exposure, male offspring showed arsenic-induced dose-related increases in hepatocellular carcinoma incidence and multiplicity, as well as increases in adrenal tumor incidence and multiplicity. In female offspring, an increase in epithelial ovarian tumors occurred with arsenic exposure regardless of TPA exposure. Females also showed pre-neoplastic lesions of the reproductive tract, including hyperplasia of the uterus and oviduct, after arsenic but independent of TPA exposure. Although TPA had no effect on skin tumors, it promoted arsenic initiated liver tumors in females and lung tumors in both sexes. Thus, inorganic arsenic, as a single agent, can consistently act as a complete transplacental carcinogen in mice, inducing tumors at multiple sites, and as a tumor initiator in some tissues. Skin tumors were not initiated by arsenic in mouse fetuses possibly indicating tissue-specific mechanisms of action. This study indicates that gestation is a period of high sensitivity to arsenic carcinogenesis.