Differential effect of gestation stage on benzo(a)pyrene-induced micronucleus formation and/or covalent DNA modifications in mice

Benzo(a)pyrene (BP), an environmental carcinogen, binds ubiquitously to the DNA of maternal and fetal tissues (Lu et al., Cancer Res., 46: 3046-3054, 1986). These studies further investigated the effect of gestation age on the induction of genetic damage by BP. Timed-pregnant ICR mice were treated with one dose of BP on various days of gestation and sacrificed 24-120 h after treatment. At the molecular level, BP covalently bound to the DNA of adult bone marrow and fetal liver of mice at all gestation ages. Compared to the nonpregnant mice (adduct level = 15 adducts/10(8) bases), the adduct levels in the pregnant adult bone marrow were decreased up to 50% during early gestation (days 3-9) and then increased steadily to a 4-fold excess over nonpregnant values during late gestation (days 15-18). In the fetal liver, adduct levels exhibited little variation (3-4 adducts/10(8) bases) between days 11 and 15 of gestation and then increased sharply to 14 adducts/10(8) bases after day 16. At the cellular level, a higher percentage of polychromatic RBCs from adult and fetal mice after BP treatment contained micronuclei (MN) than controls (solvent or untreated). Bone marrow from pregnant mice exhibited greater increases in the formation of MN during early gestation (days 3-9) relative to late gestation (days 15-18), compared to the nonpregnant mice. In the fetuses, the amounts of MN formed were higher than those found in the adult nonpregnant or maternal mice, but these amounts decreased with gestation progression. Thus, MN induction with gestation progression differed from DNA adduction in adults and fetuses. In addition, the dose and time responses of MN formation also differed from those of covalent DNA modifications, when analyzed in the bone marrow of pregnant mice treated on gestation day 5. Collectively, our results showed that pregnancy and development modulate different types of genetic damage in different ways. Fetal tissues may be more sensitive than maternal tissues to genetic damage. Factors in addition to DNA adduct formation may be responsible for MN induction.     

Detection of in vivo DNA repair synthesis in mouse liver and lung induced by treatment with benzo(a)pyrene or 4-nitroquinoline 1-oxide

We examined in vivo DNA repair synthesis in liver and lung of A/HeJ mice treated with benzo(a)pyrene (BP) or 4-nitroquinoline 1-oxide. To differentiate between the removal of carcinogen metabolite:DNA adducts due to cell turnover and DNA repair, we measured unscheduled DNA synthesis (UDS) in the nonreplicating DNA fraction. Mice were exposed to bromodeoxyuridine pellets 1 hr prior to carcinogen treatment. Immediately following carcinogen exposure, mice received 4 hourly i.v. doses of [3H]thymidine. Mice were sacrificed 5 hr post-carcinogen treatment, and DNA was isolated. Purified DNA was then separated into newly replicated and nonreplicated DNA by ultracentrifugation in alkaline CsCl gradients. BP induced UDS in the liver at p.o. doses of 0.3 and 3.0 mg/mouse, whereas we failed to detect UDS in the lung. However, 4-nitroquinoline 1-oxide, another lung carcinogen, induced a definite repair response in the lung but not in the liver. It is not clear why mouse lung cells have the capacity to repair 4-nitroquinoline 1-oxide-induced damage to DNA and not the damage induced by BP, since both of these lung carcinogens form bulky adducts with DNA. These results demonstrate that (a) the in vivo disappearance of BP metabolite:DNA adducts from the lung of the A/HeJ mouse is due to cell turnover, whereas the disappearance of adducts from the liver is due, in part, to DNA repair and (b) induction of in vivo UDS after treatment with two different lung carcinogens is both tissue and carcinogen dependent in this mouse strain.     

Cisplatin-DNA adduct formation in maternal and fetal rat tissues after transplacental cisplatin exposure

Cis-diamminedichloroplatinum (II) (cisplatin), given to pregnantrats at 5 mg/kg body weight (bw) is a transplacental carcinogenfor fetal liver, kidney, nervous system and lung, resultingin tumor incidences of 22.5, 10.5, 6.1 and 7.5% respectively,in offspring grown to adulthood (B.A. Diwan et al., 1995, Toxicol.Appl. Pharm., 132, 115). In this study, the capacity of cisplatinto pass through the placental barrier and bind convalently toDNA in maternal and fetal tissues was evaluated. Pregnant F344/NOrats were injected i.p. with single does of 5, 10 or 15 mg cisplatin/kgbw at 18 days of gestation and sacrificed 24 h later. Cisplatin-DNAadducts were determined by dissociation-enhanced lanthanidefluroroimmunoassay (DELFIA) using both High (90 pmol/µgDNA) and low (0.50 pmol/µg DNA) Modified cisplatin-DNAstandards and atomic absorbance spectrometry (AAS). The adductquantities determined by the two DELFIAs varied in concert,but the DELFIA with Low Modified standard gave actual valuessimilar to those observed with AAS. In maternal and fetal tissues,with the exception of placenta in one experiment and maternalkidney in another experiment, the extent of cisplatin-DNA adductformation increased with dose. In maternal kidney, the low adductlevels observed at the 15 mg/kg dose may reflect kidney toxicity.Fetal kidney, liver and lung contained fewer cisplatin-DNA adductsthan the corresponding maternal tissues. In contrast, at 5 and15 mg/kg, fetal brain DNA contained higher adduct levels thanmaternal brain DNA. This study demonstrates the presence ofDNA damage induced by cisplatin in multiple maternal and fetalrat tissues at tumorigenic doses of drug; the results are thereforeconsistent with the hypothesis that genotoxic mechanisms playan important role in the drug-induced tumor incidence.     

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.     

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.     

Elevated 8-hydroxy-2'-deoxyguanosine levels in lung DNA of A/J mice and F344 rats treated with 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone and inhibition by dietary 1,4-phenylenebis(methylene)selenocyanate

1,4-Phenylenebis(methylene)selenocyanate (p-XSC) is an effective chemopreventive agent against 4-(methylnitrosamino)-1-(3-pyridyl)-1- butanone (NNK)-induced lung adenoma in female A/J mice. While p-XSC can effectively inhibit NNK-induced DNA methylation in female A/J mice and in male F344 rats, its effect on NNK-induced oxidative DNA damage had not been determined. Thus, the effect of p-XSC on the levels of 8- hydroxy-2'-deoxyguanosine (8-OH-dG) in lung DNA from A/J mice and F344 rats treated with NNK was examined. Mice were given NNK by gavage (0.5 mg/mouse in 0.2 ml corn oil, three times per week for 3 weeks) or by a single i.p. injection (2 mg/mouse in 0.1 ml saline) while maintained on a control diet (AIN-76A) or control diet containing p-XSC at 10 or 15 p.p.m. (as Se) starting 1 week before NNK administration and continuing until termination. Mice were killed 2 h after the last NNK gavage in the multiple administration protocol or 2 h after the single i.p. injection. Treatment with NNK by gavage significantly elevated the levels of 8-OH-dG in lung DNA of A/J mice from 0.7 +/- 0.1 to 1.6 +/- 0.2 adducts/10(5) 2'-deoxyguanosine (dG) (P < 0.001), while dietary p- XSC (at 10 p.p.m. Se) prevented significant elevation of the levels of this lesion caused by NNK, keeping them at 0.9 +/- 0.1 adducts/10(5) dG (P < 0.003). Injection of NNK in saline also significantly increased the levels of 8-OH-dG in lung DNA of A/J mice from 1.2 +/- 0.6 to 3.6 +/- 0.8/10(5) dG adducts (P < 0.01), while dietary p-XSC (at 15 p.p.m. Se) kept these levels at 1.9 +/- 0.5 adducts/10(5) dG (P < 0.03). Rats were given a single i.p. injection of NNK (100 mg/kg body wt) in saline while being maintained on control diet (AIN-76A) or control diet containing p-XSC (15 p.p.m. as Se) starting 1 week before NNK administration and continuing until termination. The rats were killed 2 h after injection. Treatment with NNK using this protocol significantly elevated the levels of 8-OH-dG in lung DNA of F344 rats from 2.6 +/- 0.5 to 3.5 +/- 0.5 adducts/10(5) dG (P < 0.03), while dietary p-XSC (at 15 p.p.m. Se) kept the levels of this lesion at 2.2 +/- 0.6 adducts/10(5) dG (P < 0.01). Our findings suggest that the chemopreventive efficacy of p-XSC against NNK-induced lung tumorigenesis in A/J mice and F344 rats may be due in part to inhibition of oxidative DNA damage.      

Modulation of benzo[a]pyrene-induced covalent DNA modifications in adult and fetal mouse tissues by gestation stage

In these studies, we investigated the influence of gestation age on the induction of covalent DNA modifications by benzo[a]pyrene (B[a]P). Timed-pregnant ICR mice were given a single treatment of B[a]P (80 mg/kg, p.o.) on different days of gestation, killed 24 h later and analyzed for the presence of B[a]P-induced DNA adducts using the P1 nuclease version of the 32P-postlabeling method. Our results showed that B[a]P bound to embryonic, placental, fetal and maternal DNA throughout gestation with gestation-stage dependency. Overall, B[a]P bound less to maternal DNA during organogenesis and placentation compared to other stages of gestation and to the non-pregnant stage. The ontogenesis of B[a]P-induced DNA adducts in fetal tissues exhibited organ specificity that had two different types of profiles. With advancing gestation age, one type (lung, carcass and placenta) exhibited a steady linear increase, and the other type [gastrointestinal tract (GIT) and skin] a biphasic increase. In the fetal and maternal organs, adduct levels peaked 2 days before parturition. Over the course of gestation, fetal adduct levels were 70-100% of adult levels in the skin, 7-12% in the GIT, 25-40% in the liver and 15-80% in the lung. The adduct levels in many fetal organs exhibited little relationship to placental adduct levels throughout gestation. Collectively, our results indicate that: (i) transplacental DNA damage induced by B[a]P is determined mainly by fetal competence in metabolic activation and/or detoxification of B[a]P; and (ii) events occurring during placentation and organogenesis inhibit B[a]P binding to maternal tissues.     

Placental transfer of 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone instilled intratracheally in Syrian golden hamsters.

The tobacco-specific N-nitrosamine 4-(methylnitrosamino)-1-(3-pyridyl)-1- butanone (NNK) is a potent tumorigen in adult Syrian golden hamsters and an active transplacental carcinogen in this species. In this study, we have investigated the biodistribution and metabolism of NNK in maternal and fetal hamster tissues as a function of the dose and the time after NNK treatment. Hamsters on day 15 of gestation were instilled intratracheally with single doses (0.05-100 mg/kg) of [5-3H]NNK and sacrificed 30 min later or treated with a single dose (25 mg/kg) of [5-3H]NNK and sacrificed at various times (5-360 min) after treatment. Total radioactivity was quantified in maternal tissues (liver, lung, kidney, placenta, and stomach), in whole fetus and in fetal tissues (liver and lung). NNK and its metabolites were extracted from selected tissues (maternal plasma, amniotic fluid, fetal liver, and lung) and assayed by high-performance liquid chromatography-scintigraphy. Thirty min after treatment, radioactivity associated with NNK and its metabolites showed similar widespread tissue distribution patterns at all doses, with a linear dose relationship observed in whole fetus and fetal tissues. NNK levels detected in maternal plasma, amniotic fluid, fetal liver, and lung were also related linearly to dose. At high doses (25 mg/kg or more) of NNK, 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanol was the major metabolite detected in maternal plasma. Pyridine N-oxidation of NNK predominated at the lowest doses (0.05 and 0.5 mg NNK/kg). The toxicokinetics of NNK demonstrated that this carcinogen is rapidly absorbed from the maternal lung (less than 5 min), metabolized mainly to 4-(methylnitrosamino)-1- (3-pyridyl)-1-butanol, and quickly distributed into the maternal-fetal compartment. Both NNK and its main metabolite 4-(methylnitrosamino)-1-(3- pyridyl)-1-butanol were eliminated slowly from the amniotic fluid, with levels still detectable up to 6 h after NNK treatment. These results demonstrated that NNK instilled intratracheally in pregnant hamsters crossed the placental barrier even at low doses. Moreover, NNK quickly reached fetal tissues and amniotic fluid and was eliminated slowly from these tissues, resulting in an extended exposure of the fetus to this tobacco-specific carcinogen.     

Ethylnitrosourea-induced transplacental carcinogenesis in the mouse: tumor response, DNA binding, and adduct formation

We have confirmed previous results which suggest that transplacental exposure of fetal mice to carcinogens does not cause an increase in tumor incidence as they mature unless treatment occurs after midorganogenesis. In C3HeB/FeJ mice we found a negligible increase in tumor incidence and multiplicity following transplacental exposure to the direct-acting carcinogen ethylnitrosourea (ENU) on gestation day 10, but significant increases in lung and liver tumor incidence following exposure on days 13 or 15 or in adults. To explore the possibility that this observed difference is due to differences in the biodistribution of the carcinogen or its interaction with cellular macromolecules, the level of covalent binding between ENU and fetal and maternal DNA following an i.p. injection of a dose of 50 mg/kg of tritium-labeled ENU was measured 30 min after its injection into pregnant females on days 10, 13, and 15 of gestation. The DNA from fetal and maternal lung, liver, and brain was isolated and the amount of covalent binding estimated from the dpm/mg DNA recovered. Samples of DNA were hydrolyzed and chromatographed to determine that the bound tritium was associated with ENU-DNA adducts and not as a product of DNA synthesis. The level of binding of ENU to fetal DNA was equivalent at all gestation days studied but was significantly less than maternal tissues. Binding to the DNA of maternal liver was 4-fold greater than to fetal DNA while maternal lung and brain DNA were bound at intermediate levels. We conclude that the lack of carcinogenic response to ENU documented here, in fetal mice exposed early in gestation (day 10), is not due to differences in ENU binding to fetal DNA during development.     

Benzo(a)pyrene diol epoxide-I-DNA adduct formation in the epidermis and lung of SENCAR mice following topical application of crude coal tar

The levels of benzo[a]pyrene diol epoxide-I-deoxyguanosine (BPDE-I-dG) adduct formation in epidermis and lung of SENCAR mice following the topical application of benzo[a]pyrene (BP) alone, crude coal tar (CCT) alone, and the two combined were determined in an enzyme linked immunosorbent (ELISA) assay using monoclonal antibodies. Topical application of two doses of BP (20 micrograms) at 72-h intervals, with sacrifice 24 h later resulted in the formation of 197 fmol and 205 fmol BPDE-I-dG adducts per mg DNA in epidermis and lung, respectively. Topical application of 0.5 ml CCT alone resulted in the formation of 278 fmol and 410 fmol BPDE-I-dG adducts per mg DNA in epidermis and lung, respectively. Simultaneous topical application of 20 micrograms BP and CCT (0.1-0.5 ml) resulted in substantially lower BPDE-I-dG adducts in the epidermis as well as in the lung. Our results suggest that CCT may contain inhibitors of carcinogen-DNA adduct formation and that topical application of CCT produces greater effects on DNA-adduct formation in lung than in epidermis. Thus the cancer-causing potency of the polycyclic aromatic hydrocarbons (PAHs) in CCT may be reduced by other anticarcinogenic constituents present in CCT and systemic absorption of carcinogenic PAHs in CCT applied to skin might have tumorigenic effects in other tissues.     

Tissue distribution and macromolecular binding of extremely low doses of [14C]-benzene in B6C3F1 mice

The tissue distribution and macromolecular binding of benzene was studied over a dose range spanning nine-orders of magnitude to determine the nature of the dose-response and to establish benzene's internal dosimetry at doses encompassing human environmental exposures. [14C]-Benzene was administered to B6C3F1 male mice at doses ranging between 700 pg/kg and 500 mg/kg body wt. Tissues, DNA and protein were analyzed for [14C]-benzene content between 0 and 48 h post-exposure (625 Ng/kg and 5 microg/kg dose) by accelerator mass spectrometry (AMS). [14C]-Benzene levels were highest in the liver and peaked within 0.5 h of exposure. Liver DNA adduct levels peaked at 0.5 h, in contrast to bone marrow DNA adduct levels, which peaked at 12-24 h. Dose- response assessments at 1 h showed that adducts and tissue available doses increased linearly with administered dose up to doses of 16 mg/kg body wt. Tissue available doses and liver protein adducts plateau above the 16 mg/kg dose. Furthermore, a larger percentage of the available dose in bone marrow bound to DNA relative to liver. Protein adduct levels were 9- to 43-fold greater than DNA adduct levels. These data show that benzene is bioavailable at human-relevant doses and that DNA and protein adduct formation is linear with dose over a dose range spanning eight orders of magnitude. Finally, these data show that the dose of bioactive metabolites is greater to the bone marrow than the liver and suggests that protein adducts may contribute to benzene's hematoxicity.      

Elevated mitochondrial cisplatin-DNA adduct levels in rat tissues after transplacental cisplatin exposure

Although there is evidence that the toxic effects of cis- diamminedichloroplatinum(II) (cisplatin) include morphologically abnormal mitochondria, direct demonstrations of mitochondrial DNA damage by this chemotherapeutic agent have rarely been reported. Here we show that, in rats exposed to a single dose of cisplatin during gestation, cisplatin-DNA binding levels in both maternal and fetal liver and brain mitochondrial DNA are higher than those observed in genomic DNA. Pregnant F344/NCr rats were injected i.p. with either 5 or 15 mg cisplatin/kg body wt at 18 days of gestation and killed 24 h later. Cisplatin-DNA adducts were determined by dissociation-enhanced lanthanide fluoroimmunoassay using a cisplatin-DNA standard modified in the same range as the biological samples. Values for genomic cisplatin- DNA adducts in multiple maternal and fetal tissues have been presented elsewhere. Here, genomic DNA adduct levels for liver, brain, kidney and placenta are reported again for comparison with mitochondrial DNA adduct levels in the same tissues. In maternal and fetal brain, mitochondrial DNA adduct levels were approximately 7- to 50-fold higher than genomic DNA adduct levels, and in fetal liver they were approximately 2- to 16-fold higher than genomic DNA adduct levels. These studies demonstrate extensive cisplatin-DNA adduct formation in brain and liver mitochondria of fetal rats exposed transplacentally and suggest that mitochondrial DNA in some organs may be a particular target for cisplatin genotoxicity.      

Lung tumor induction in strain A mice with benzotrichloride

Benzotrichloride (BTC) is used in the synthesis of benzoyl chloride and benzoyl peroxide. Epidemiological data suggest that BTC is a human lung carcinogen. In the present study, BTC was evaluated for its ability to induce lung adenomas in strain A/J mice. Four groups of 15 male and 15 female A/J mice were injected i.p. with either tricaprylin or BTC in tricaprylin three times a week for 8 weeks. BTC groups received doses totaling 1440 mg/kg, 719 mg/kg or 287 mg/kg. The mean number of lung tumors per mouse was 127 87 +/- 5.81, 43 +/- 2.44, and 17.73 +/- 1.09 in the groups treated with either 1440 mg/kg, 719 mg/kg, or 287 mg/kg, respectively. Tricaprylin-vehicle controls had a mean number of 0.46 +/- 0.15 lung tumors per mouse. Therefore, BTC produced a significant (P less than 0.001) and dose-related increase in the lung tumor response when compared to tricaprylin controls and is a potent carcinogen in the strain A mouse lung tumor bioassay.     

Preclinical relevance of dosing time for the therapeutic index of gemcitabine-cisplatin

The relevance of gemcitabine timing for chronotherapeutic optimisation was investigated. Healthy mice received multiple doses of gemcitabine (120, 160 or 200 mg kg(-1) injection (inj)(-1)) at one of six circadian times (3, 7, 11, 15, 19 or 23 h after light onset--HALO) on days 1, 4, 7 and 10 or a single dose of gemcitabine (400 mg kg(-1)) at 11 or 23 HALO+/-cisplatin (5 mg kg(-1) at 1 min, 4 or 8 h later). Mice bearing Glasgow osteosarcoma received multiple doses of gemcitabine (200 mg kg(-1) inj(-1)) at 11 or 23 HALO+/-cisplatin (5 mg kg(-1) inj(-1) at 1 min or 4 h later) on days of 10, 13, 16 and 19 following tumour inoculation. A circadian rhythm in body weight loss was statistically validated, with 1030 HALO corresponding to the least toxic time (95% CL, 0800 to 1300). Gemcitabine dosing produced least body weight loss and least neutropenia after injection at 11 vs 23 HALO, whether the drug was given alone or with cisplatin (P=0.001). Gemcitabine-cisplatin tolerability was improved by dosing gemcitabine at 11 HALO and CDDP at 15 HALO (P<0.001). The administration of this schedule to tumour-bearing mice increased median survival three-fold as compared to treatments where both drugs were given simultaneously at 11 or 23 HALO (P=0.02). The optimal schedule would correspond to the delivery of gemcitabine upon awakening and cisplatin near mid-activity in cancer patients.     

Pharmacokinetics and tissue distribution of halofuginone (NSC 713205) in CD2F1 mice and Fischer 344 rats

PURPOSE: Halofuginone (HF) inhibits synthesis of collagen type I and matrix metalloproteinase-2 and is being considered for clinical evaluation as an antineoplastic agent. Pharmacokinetic studies were performed in rodents to define the plasma pharmacokinetics, tissue distribution, and urinary excretion of HF after i.v. delivery and the bioavailability of HF after i.p. and oral delivery. MATERIALS AND METHODS: Studies were performed in CD2F1 mice and Fischer 344 rats. In preliminary toxicity studies in mice single HF i.v. bolus doses between 1.0 and 5.0 mg/kg were used. Pharmacokinetic studies were conducted in mice after administration of 1.5 mg/kg HF. In preliminary toxicity studies in male rats HF i.v. bolus doses between 0.75 and 4.5 mg/kg were used. In pharmacokinetic studies in rats an HF dose of 3.0 mg/kg was used. Compartmental and non-compartmental analyses were applied to the plasma concentration versus time data. Plasma, red blood cells, various organs, and urine were collected for analysis. RESULTS: HF doses > or = 1.5 mg/kg proved excessively toxic to mice. In mice, i.v. bolus delivery of 1.5 mg/kg HF produced "peak" plasma HF concentrations between 313 and 386 ng/ml, and an AUC of 19,874 ng/ml min, which corresponded to a total body clearance (CLtb) of 75 ml/min per kg. Plasma HF concentration versus time data were best fit by a two-compartment open linear model. The bioavailability of HF after i.p. and oral delivery to mice was 100% and 0%, respectively. After i.v. bolus delivery to mice, HF distributed rapidly to all tissues, except brain. HF persisted in lung, liver, kidney, spleen, and skeletal muscle longer than in plasma. In the oral study, HF was undetectable in plasma and red blood cells, but was easily detectable in kidney, liver, and lung, and persisted in those tissues for 48 h. Urinary excretion of HF accounted for 7-11% of the administered dose within the first 72 h after i.v. dosing and 15-16% and 16% of the administered dose within 24 and 48 h, respectively, after oral dosing. There were no observed metabolites of HF in mouse plasma or tissues. In rats, i.v. bolus delivery of 3.0 mg/kg produced a "peak" plasma HF concentration of 348 ng/ml, and an AUC of 43,946 ng/ml min, which corresponded to a CLtb of 68 ml/min per kg. Plasma HF concentration versus time data were best fit by a two-compartment open linear model. After i.v. bolus delivery to rats, HF distributed rapidly to all tissues, with low concentrations detectable in brain and testes. HF was detectable in some tissues for up to 48 h. HF could be detected in rat plasma after a 3 mg/kg oral dose. Peak HF concentration (34 ng/ml) occurred at 90 min, but HF concentrations were less than the lower limit of quantitation (LLQ) by 420 min. Urinary excretion of HF accounted for 8-11% of the administered dose within the first 48 h after i.v. dosing. No HF metabolites were detected in plasma, tissue, or urine. CONCLUSIONS: HF was rapidly and widely distributed to rodent tissues and was not converted to detectable metabolites. In mice, HF was 100% bioavailable when given i.p. but could not be detected in plasma after oral administration, suggesting limited oral bioavailability. However, substantial concentrations were present in liver, kidney, and lungs. HF was present in rat plasma after an oral dose, but the time course and low concentrations achieved precluded reliable estimation of bioavailability. These data may assist in designing and interpreting additional preclinical and clinical studies of HF.     

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.     

Fate of the tobacco-specific carcinogen 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone in pregnant and newborn C57BL mice

Whole-body autoradiography of pregnant C57Bl mice injected intravenously with the tobacco-specific N-nitrosamine, 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK), indicated that NNK and/or its metabolites can diffuse through the placenta and reach the fetal tissues. During the last days of gestation, nasal, pulmonary and hepatic tissues develop the enzymatic capacity to activate NNK to alkylating species which bind covalently to cellular macromolecules. Within 4 h of the injection, a considerable proportion of NNK metabolites present in the fetal tissues are excreted in the amniotic fluid via the fetal urinary tract. Incubation of tissue slices with NNK indicated that the nose, the lung and the liver of 13-day-old fetuses could reduce NNK to 4-(methylnitrosamino)-1-(3-pyridyl)butan -1-ol (NNA1), but could not activate NNK by alpha-carbon hydroxylation. However, these activating enzymes were competent in 18-day old fetuses, and the activities increased during the first six days of life. The results provide evidence that NNK could exert genotoxic effects transplacentally and in newborn mice.     

Tumor multiplicity, DNA adducts and K-ras mutation pattern of 5-methylchrysene in strain A/J mouse lung

This study was undertaken to evaluate the carcinogenic potenlialof 5-methylchrysene (5-MeC) in strain A/J mouse lung and tocorrelate the 5-MeC-DNA adduct profile in lung tissue with themutation spectrum in the K-ras gene of lung tumors. Strain A/Jmice received a single i.p. injection of 5-MeC at doses of 10,50, 100 and 200 mg/kg and after 24, 48 and 72 h their lungswere collected for DNA adduct analysis. Eight months later,lungs from the remaining mice were harvested and the lung tumorscounted and collected for subsequent mutational analysis ofthe K-ras gene. 5-MeC was found to be a potent lung carcinogenin strain A/J mice, inducing more than 100 tumors/mouse at aconcentration of 200 mg/kg. Six 5-MeC-DNA adducts were observed;one adduct comigrated with the standard N²-deoxyguanosine adductof 5-MeC-diol-epoxide I [1R,2S,3S-trihydroxy-4R-(N²-deoxyguanosyl-3'-phosphate)-1,2,3,4-tetrahydro-5-methyl-chrysene],derived from the bay-region diol-epoxide of 5-MeC. DNAs isolatedfrom 5-MeC-induced lung tumors were evaluated for activatingmutations in the K-ras gene by polymerase chain reaction-singlestrand conformation polymorphism and direct DNA sequencing analysis.Mutations were detected in 44 of 49 (90%) 5-MeC-induced tumorsand the mutations were GGT     

Reduced lung tumorigenesis in human methylguanine DNA--methyltransferase transgenic mice achieved by expression of transgene within the target cell

Human methylguanine-DNA methyltransferase (MGMT) transgenic mice expressing high levels of O6-alkylguanine-DNA alkyltransferase (AGT) in lung were crossbred to A/J mice that are susceptible to pulmonary adenoma to study the impact of O6-methylguanine (O6mG)-DNA adduct repair on NNK-induced lung tumorigenesis. Expression of the chimeric human MGMT transgene in lung was identified by northern and western blot analysis, immunohistochemistry assay and enzymatic assay. AGT activity was 17.6 +/- 3.2 versus 1.2 +/- 0.4 fmol/microg DNA in lung of MGMT transgenic mice compared with non-transgenic mice. Immunohistochemical staining with anti-human AGT antibody showed that human AGT was expressed throughout the lung. However, some epithelial cells of bronchi and alveoli did not stain for human AGT, suggesting that the human MGMT transgene expression was heterogeneous. After 100 mg/kg NNK i.p. injection in MGMT transgenic mice, lung AGT activity remained much higher and levels of lung O6mG-DNA adducts in MGMT transgenic mice were lower than those of non-transgenic mice. In the tumorigenesis study, mice received 100 mg/kg NNK at 6 weeks of age and were killed 44 weeks later. Ten of 17 MGMT transgenic mice compared with 16 of 17 non-transgenic mice had lung tumors, P < 0.05. MGMT transgenic mice had lower multiplicity and smaller sized lung tumors than non-transgenic mice. Moreover, a reduction in the frequency of K-ras mutations in lung tumors was found in MGMT transgenic mice (6.7 versus 50% in non-transgenic mice). These results indicate that high levels of AGT expressed in mouse lung reduce lung tissue susceptibility to NNK-induced tumorigenesis due to increased repair capacity for O6mG, subsequently, decreased mutational activation of K-ras oncogene. Heterogeneity in the level of AGT expressed in different lung cell populations or other forms of carcinogenic DNA damage caused by NNK may explain the residual incidence of lung tumors in MGMT transgenic mice.