BaP-DNA Adducts Research Articles

Modulation of adult rat benzo(a)pyrene (BaP) metabolism and DNA adduct formation by neonatal diethylstilbestrol (DES) exposure

This study seeks to elucidate the role of diethylstilbestrol (DES), a synthetic estrogen on benzo(a)pyrene (BaP) metabolism in the male rat reproductive tissues. Offspring of timed-pregnant Sprague-Dawley rats were neonatally treated on days 2, 4, and 6 post-partum with 1.45 μmol/kg of DES. Ten weeks after birth, the adult rats were challenged with radiolabeled benzo(a)pyrene ( 3H BaP) (10 μmol/kg) and the rats were sacrificed 2 h after BaP exposure. Prostrate, testis, lung, liver, urine and feces samples were collected and extracted using a mixture of H 2O, MeOH and CHCl 3. The extracts were analyzed by reverse phase HPLC. The concentrations of BaP organic metabolites in DES rats were lower compared to controls (vehicle-treated rats). On the other hand, concentrations of aqueous metabolites were significantly increased in DES treated animals. The toxication to detoxication ratios were significantly decreased in DES rats compared to controls. This trend is also reflected in the decreased concentrations of BaP-DNA adducts in DES rats. Collectively these results suggest that DES is capable of modulating the metabolic pathway of BaP towards detoxification thereby preventing the manifestation of toxicity.

Oral exposure to benzo[a]pyrene in the mouse: detoxication by inducible cytochrome P450 is more important than metabolic activation.

The cytochrome P450 (CYP1A1) enzyme metabolically activates many polycyclic aromatic hydrocarbons, including benzo[a]pyrene (BaP), to DNA- and protein-binding intermediates that are associated with toxicity, mutagenesis, and carcinogenesis. As a result, it is widely accepted that CYP1A1 potentiates the toxicity of this class of chemicals. In distinct contrast, we show here that CYP1A1 inducibility is essential in the detoxication of oral BaP. We compared Cyp1a1(-/-) knockout mice, having the genetic absence of the CYP1A1 enzyme, with Cyp1a1(+/+) wild-type mice. At an oral BaP dose of 125 mg/kg/day, Cyp1a1(-/-) mice died within 30 days whereas Cyp1a1(+/+) mice displayed no outward signs of toxicity. The rate of BaP clearance was 4-fold slower in Cyp1a1(-/-) than Cyp1a1(+/+) mice. The cause of death in Cyp1a1(-/-) mice receiving oral BaP seemed to be immunotoxicity, including toxic chemical depression of the bone marrow; some toxic effects in Cyp1a1(-/-) mice were noted at a BaP dose as low as 1.25 mg/kg/day. DNA post-labeling studies demonstrated dramatically higher BaP-DNA adduct levels in all Cyp1a1(-/-) tissues assayed, with the exception of the small intestine, which is probably a major site of BaP metabolism in Cyp1a1(+/+) mice. Different BaP-DNA adduct patterns were also observed between the two genotypes receiving oral BaP. Despite previous studies in vitro and in cell culture that have shown a participatory role for CYP1A1 in BaP toxicity, the present data indicate that, in the intact animal, inducible CYP1A1 is extremely important in detoxication and protection against oral BaP toxicity.

Molecular evidence of an interaction between prenatal environmental exposures and birth outcomes in a multiethnic population.

Inner-city, minority populations are high-risk groups for adverse birth outcomes and also are more likely to be exposed to environmental contaminants, including environmental tobacco smoke (ETS), benzo[a]pyrene (BaP), and other polycyclic aromatic hydrocarbons (PAHs) found in urban air. In a sample of nonsmoking African-American and Dominican women, we evaluated the effects on birth outcomes of prenatal exposure to ETS, using questionnaire data and plasma cotinine as a biomarker of exposure, and environmental PAHs using BaP-DNA adducts as a molecular dosimeter. We previously reported that among African Americans, high prenatal exposure to PAHs estimated by prenatal personal air monitoring was associated with lower birth weight (p = 0.003) and smaller head circumference (p = 0.01) after adjusting for potential confounders. In the present analysis, self-reported ETS was associated with decreased head circumference (p = 0.04). BaP-DNA adducts were not correlated with ETS or dietary PAHs. There was no main effect of BaP-DNA adducts on birth outcomes. However, there was a significant interaction between the two pollutants such that the combined exposure to high ETS and high adducts had a significant multiplicative effect on birth weight (p = 0.04) and head circumference (p = 0.01) after adjusting for ethnicity, sex of newborns, maternal body mass index, dietary PAHs, and gestational age. This study provides evidence that combined exposure to environmental pollutants at levels currently encountered in New York City adversely affects fetal development.

Biomarkers in maternal and newborn blood indicate heightened fetal susceptibility to procarcinogenic DNA damage.

Polycyclic aromatic hydrocarbons (PAHs) such as benzo[a]pyrene (BaP) are widespread air contaminants released by transportation vehicles, power generation, and other combustion sources. Experimental evidence indicates that the developing fetus is more susceptible than the adult to carcinogenic effects of PAHs, although laboratory studies in rodents suggest that the dose to fetal tissues is an order of magnitude lower than that to maternal tissues. To assess fetal versus adult susceptibility to PAHs and environmental tobacco smoke (ETS), we compared carcinogen-DNA adducts (a biomarker associated with increased cancer risk) and cotinine (a biomarker of tobacco smoke exposure) in paired blood samples collected from mothers and newborns in New York City. We enrolled 265 nonsmoker African-American and Latina mother–newborn pairs in New York City between 1997 and 2001 (estimated average ambient air BaP concentrations < 0.5 ng/m3). Despite the estimated 10-fold lower fetal dose, mean levels of BaP-DNA adducts as determined by high-performance liquid chromatography–fluorescence were comparable in paired New York City newborn and maternal samples (0.24 adducts per 108 nucleotides, 45% of newborns with detectable adducts vs. 0.22 per 108 nucleotides, 41% of mothers with detectable adducts). However, by the Wilcoxon signed-rank test, the levels in newborns were higher (p = 0.02). Mean cotinine was higher in newborns than in mothers (1.7 ng/mL, 47% detectable vs. 1.28 ng/mL, 44% detectable). Consistent with our prior study in a Caucasian Polish population, these results indicate increased susceptibility of the fetus to DNA damage and reduced ability to clear ETS constituents. The findings have implications for risk assessment, given the need to protect children as a sensitive subset of the population.

Effect of arsenic on benzo[a]pyrene DNA adduct levels in mouse skin and lung.

Concomitant exposures to arsenic and polycyclic aromatic hydrocarbons (PAHs) such as benzo[a]pyrene (BaP) are widespread. While BaP acts by binding to and inducing mutations in critical sites on DNA, the mechanism(s) of arsenic carcinogenesis remains unknown. Data from epidemiological studies of arsenic copper smelter workers and arsenic ingestion in drinking water suggest a positive interaction for arsenic exposure and smoking and lung cancer. A previous in vitro study showed that arsenic potentiated the formation of DNA adducts at low doses of BaP and arsenic. The present study was conducted to test the effect of arsenic on BaP-DNA adduct formation in vivo. We hypothesized that arsenic co-treatment would significantly increase BaP adduct levels in C57BL/6 mouse target organs: skin and lung. Treatment groups were: five mice, -BaP/-arsenic; five mice, -BaP/+arsenic; 15 mice, +BaP/-arsenic; 15 mice, +BaP/+arsenic. Mice in the appropriate groups were provided sodium arsenite in drinking water (2.1 mg/l), ad libitum, for 13 days (starting 9 days before BaP treatment), and 200 nmol BaP/25 ml acetone (or acetone alone) was applied topically, once per day for 4 days. DNA was extracted from skin and lung and assayed by (32)P-postlabeling. Statistical comparisons were made using independent t-tests (unequal variances assumed). BaP-DNA adduct levels in the +BaP groups were significantly higher than -BaP controls. Arsenic co-treatment increased average BaP adduct levels in both lung and skin; the increase was statistically significant in the lung (P = 0.038). BaP adduct levels in the skin of individual animals were positively related to skin arsenic concentrations. These results corroborate our in vitro findings and provide a tentative explanation for arsenic and PAH interactions in lung carcinogenesis.

The combination of benzo[a]pyrene and ultraviolet A causes an in vivo time-related accumulation of DNA damage in mouse skin.

Polycyclic aromatic hydrocarbons, including benzo[a]pyrene (BaP), are ubiquitous environmental carcinogens. BaP is metabolized in vivo to reactive intermediates that become covalently bound to DNA and form BaP-DNA adducts, an initial event in carcinogenesis. Ultraviolet A (UVA) synergizes with BaP to significantly enhance genetic damage and accelerate carcinogenic processes. This study was initiated to investigate in vivo cellular changes related to carcinogenesis induced by repeated exposures to BaP plus UVA. Simulated chronic exposure to an environmental carcinogen and sunlight was conducted through biweekly topical application of BaP followed 2 h later by UVA exposure over a 10 week period. BaP diol epoxide (BPDE)-DNA adducts were measured in vivo by immunohistochemistry using an anti-BPDE-DNA monoclonal antibody. Oxidative DNA damage was measured by the detection of 8-hydroxy-2'-deoxyguanosine (8-OHdG) formation using high-performance liquid chromatography. Alterations in the cell cycle that were relevant to carcinogenesis were revealed by changes in p53, as identified in vivo using a polyclonal anti-p53 antibody. We found that cells containing BPDE-DNA adducts and nuclear p53 expression significantly increased between 2 and 10 weeks of BaP-UVA treatment, whereas neither BPDE-DNA adducts nor significant changes in p53 were observed in untreated skin. Using regression analysis, oxidative 8-OHdG damage also showed a parallel increase over 2-10 weeks (r = 0.80). These results indicate that genetic damage caused by exposures to BaP plus UVA accumulates with time and increases the potential for inductive events leading to carcinogenesis and tumor formation.

Effects of benzo[ a]pyrene exposure on a fish population resistant to the toxic effects of dioxin-like compounds

Effects of a model polycyclic aromatic hydrocarbon (PAH) were compared in populations of the estuarine fish Fundulus heteroclitus indigenous to a reference site and one highly contaminated with polychlorinated biphenyls (PCBs) and other compounds. The fish population resident to the PCB-contaminated site is genetically resistant to those PCB congeners categorized as dioxin-like compounds (DLCs) that act through the aryl hydrocarbon receptor (AHR). In response to DLC exposures, these DLC-resistant fish showed poor inducibility for enzymes known to be regulated by the AHR pathway and important for the metabolism of xenobiotics including some PAHs that also act as AHR agonists. Therefore, a laboratory study using the model PAH, benzo[ a]pyrene (BaP), was conducted to evaluate how PAHs might affect these wild fish populations that differed in their inherent sensitivities to DLCs and in their tissue concentrations of contaminants. Following BaP treatment, the activities of two xenobiotic metabolizing enzymes and the concentrations of BaP-DNA adducts, as measured using the 32P-postlabeling method, were lower in the livers of DLC-resistant than reference fish. These results suggest that DLC-resistance could provide protection following chronic exposures to PAHs from the long-term consequences of DNA adduct formation, such as cancer. Alternatively, reduced metabolism and elimination of toxic or photo-activated PAHs could have acute consequences to the health and reproduction of these DLC-resistant fish and their progeny. These fish populations provide useful models to evaluate the potential costs and benefits of genetic adaptation in wildlife populations subject to anthropogenic stress.

Antibody-based nanoprobe for measurement of a fluorescent analyte in a single cell.

We report here the application of an antibody-based nanoprobe for in situ measurements of a single cell. The nanoprobe employs antibody-based receptors targeted to a fluorescent analyte, benzopyrene tetrol (BPT), a metabolite of the carcinogen benzo[a]pyrene (BaP) and of the BaP-DNA adduct. Detection of BPT is of great biomedical interest, since this species can serve as a biomarker for monitoring DNA damage due to BaP exposure and for possible precancer diagnosis. The measurements were performed on the rat liver epithelial clone 9 cell line, which was used as the model cell system. Before making measurements, the cells were treated with BPT. Nanoprobes were inserted into individual cells, incubated 5 min to allow antigen-antibody binding, and then removed for fluorescence detection. We determined a concentration of 9.6+/-0.2x10(-11) M for BPT in the individual cells investigated. The results demonstrate the possibility of in situ measurements inside a single cell using an antibody-based nanoprobe.

EFFECT OF LOW-LEVEL SHORT-TERM o-XYLENE INHALATION ON BENZO[a]PYRENE (BaP) METABOLISM AND BaP-DNA ADDUCT FORMATION IN RAT LIVER AND LUNG MICROSOMES

o -Xylene is a commonly used solvent that alters mixed-function oxidase (MFO) activity in an organ- and isozyme-specific pattern following intraperitoneal (ip) administration. Similar MFO alterations have been observed after ip or inhalation exposure to other methyl benzenes. These MFO alterations shifted the metabolism of the carcinogen benzo[a]pyrene (BaP) toward formation of toxication metabolites in lung. The purpose of this study was to determine whether o-xylene inhalation caused similar MFO changes and whether these alterations were reflected in altered BaP metabolism and BaP-DNA adduct formation. o-Xylene (300 ppm, 6h) decreased the activity of arylhydrocarbon hydroxylase (AHH) in lung. CYP2B1 activity (benzyloxyresorufin O -dealkylase; BROD), which is responsible for metabolism of BaP to relatively nontoxic metabolites, was decreased in lung, as was, to a lesser extent, CYP1A1 (ethoxyresorufin O-dealkylase; EROD), which is responsible for metabolism of BaP to reactive/toxic metabolites. The BROD/EROD ratio, an indirect indicator of the pattern of BaP toxication/detoxication, was decreased in lung, suggesting that BaP metabolism is shifted toward toxication. No MFO alterations were observed in liver. In lung microsomes, o-xylene increased formation of 7,8-BaP-diol, while 9,10-BaP-diol, 3-OH BaP, and 9-OH BaP were decreased. In liver, o-xylene increased 9-OH BaP formation, while 4,5- and 9,10-diols as well as total diols were decreased. The toxication/detoxication ratios for BaP individual and total metabolite groups were increased in lung microsomes and unaltered in liver. The major BaP-DNA adduct, BaP diol epoxide-N 2-deoxyguanosine, was increased in lung but decreased in liver microsomes from o-xylene-exposed rats. Four minor BaP-DNA adducts were formed in lung and three in liver, only one of which (liver adduct 3) was decreased. The o-xylene-induced increase in BaP adduct formation in lung and decrease in liver indicate that coexposure to organic solvents such as the methyl benzenes may alter the carcinogenesis of BaP, or other PAHs, in an organ-specific fashion.

Comparative time-course of benzo[ a]pyrene-DNA adduct formation, and its relationship to CYP1A activity in two species of catfish

Studies have demonstrated differences between the brown bullhead ( Ameriurus nebulosus) (BB) and a closely related species of catfish, the channel catfish ( Ictalurus punctatus) (CC) in activity of important liver enzymes that may affect their ability to activate or detoxify certain procarcinogens. We have measured the formation and persistence of benzof[ a]pyrene-DNA adducts in the liver of these two species using the 32P-postlabeling (PPL) method, along with the effect of prior induction by β-naphthoflavone (βNF). In this time-course study, a total of 80 fish from each species were used. Half were induced with βNF (i.p. injections of 10 mgkg −1 at day 3 and 2) followed by a single benzo[ a]pyrene (BaP) i.p. injection (20 mg kg −1). Liver DNA and microsomes were sampled on day 1, 3, 7, 14 and 45 after the BaP dosage—adducts and ethoxyresorufin- O-deethylase (EROD) activity were analyzed. EROD activity in CC was significantly higher at most time points than in BB. In contrast, the BaP-DNA adduct profile revealed a significantly higher level of adducts in the BB than the CC. Adduct levels at most time points were significantly higher in the BB than in the CC at p < 0.05. Further characterization of the major adduct by HPLC confirmed it to be the anti-BPDE-dG adduct. The adduct profiles of BaP are in contrast to what has been observed for adducts of 2-aminoanthracene in these species, although it is consistent with the relative species susceptibility to PAH induced liver neoplasia.

The metabolic activation of tamoxifen and alpha-hydroxytamoxifen to DNA-binding species in rat hepatocytes proceeds via sulphation.

The biotransformation pathway of tamoxifen and alpha-hydroxytamoxifen to DNA-binding species was investigated in rat hepatocytes in vitro. Rat hepatocytes were isolated by in situ collagenase perfusion and then maintained in sulphate-free Dulbecco's modified Eagle's medium. Magnesium sulphate was added to the medium to give concentrations of 0-10 microM, prior to treatment for 18 h with solvent vehicle (DMSO), tamoxifen (10 microM), alpha-hydroxytamoxifen (1 microM) or benzo[a]pyrene (BaP) (10 and 50 microM). DNA was isolated and analysed by 32P-post-labelling. For tamoxifen and alpha-hydroxytamoxifen, the level of DNA adduct formation was directly proportional to the concentration of sulphate in the medium. Between 0 and 10 microM MgSO4, the DNA adduct level increased 10-fold with both compounds. Rat hepatocytes were also maintained in normal Dulbecco's modified Eagle's medium and pretreated with dehydroisoandrosterone-3-sulphate (DHEAS, a sulphotransferase inhibitor) at concentrations ranging from 0-1 mM, prior to treatment with solvent vehicle (DMSO), tamoxifen (10 microM), alpha-hydroxytamoxifen (1 microM) or BaP (50 microM). For tamoxifen and alpha-hydroxytamoxifen the level of DNA adducts was reduced to approximately one-fifth by the addition of DHEAS (0.1 mM). BaP-DNA adduct formation, which proceeds by a pathway that does not require sulphation, was not significantly affected by sulphate concentration or by addition of DHEAS, which demonstrates that the general metabolic capacity and viability of the hepatocytes were not compromised. It is concluded that the activation of tamoxifen in rat liver cells to DNA binding products proceeds predominantly through hydroxylation followed by sulphate ester formation at the alpha-position of the ethyl side chain.

Comparativein Vitroandin VivoBenzo[a]pyrene–DNA Adduct Formation and Its Relationship to CYP1A Activity in Two Species of Ictalurid Catfish

We have measured the formation and persistence ofbenzo[a]pyrene (BaP)–DNA adducts in the liver oftwo closely related species of fish, the brown bullhead(Ameriurus nebulosus) and the channel catfish(Ictalurus punctatus) using the32P-postlabeling method. Liver microsomalethoxyresorufin-O-deethylase (EROD) activity,arylhydrocarbon hydroxylase (AHH) activity, andin vitromicrosome-mediated DNA binding were all significantly higher in thechannel catfish. In anin vivotime-course experiment, fishwere either induced with βNF followed by a single BaP ip injection(20 mg/kg) or treated with corn oil. BaP–DNA adducts and EROD activityin liver were analyzed 1, 3, 7, 14, and 45 days after the BaP dosage.As in thein vitroexperiments, EROD activities in channelcatfish were significantly higher at most time points than in bullheadliver (p< 0.05). However, in contrast tothein vitrodata, the BaP–DNA adduct profile revealedsignificantly higher levels of adducts in the bullhead than the channelcatfish throughout the time course (p<0.05). Prior induction with βNF did not significantly affect thelevel or type of adduct binding to DNA in either species. Furthercharacterization of the major adduct by HPLC confirmed it to be theanti-BPDE-dGuo adduct. Analysis of tissue distribution of[14C]BaP in the two species suggested similar absorptionand initial distribution, but slower elimination from the liver ofbullhead than the catfish. The BaP–adduct profiles were consistentwith the relative species susceptibility to polycyclic aromatichydrocarbon-induced liver neoplasia. EROD activities, however, werenegatively associated with adduct levels followingin vivoexposure.

DNA Adduct Measurements in Relation to Small Intestine and Forestomach Tumor Incidence during the Chronic Feeding of Coal TAR or Benzo[A]Pyrene to Mice

Abstract In a two-year carcinogenicity bioassay, female B6C3F1 mice were fed up to 100 ppm benzo[a]pyrene (BaP) or up to 1% coal tar in the diet. Forestomach tumors were induced in mice fed BaP, with the incidence increasing sharply between 5 and 25 ppm Bap. Forestomach tumors were also observed in mice fed coal tar, with a pronounced increase at the 0.3% dose. The incidence of forestomach tumors was approximately the same at 0.3% and 0.6% coal tar, but declined at the 1.0% dose, due to early mortality from a high incidence of small intestine adenocarcinomas. DNA adduct levels were examined in the forestomachs and small intestines after feeding BaP or coal tar for 4 weeks. In BaP-treated mice, one major adduct was observed; this adduct accounted for 7-15% of the forestomach adducts in mice fed coal tar. A dose-related increase was observed in adduct levels in the forestomachs of BaP-and coal tarfed mice. The induction of forestomach tumors from BaP or coal tar was associated with the same levels of BaP-DNA adducts. In the small intestines, total adduct levels increased up to the 0.6% coal tar dose and then decreased at the 1.0% dose. Since the tumor incidence was highest at the 1.0% dose, coal tar-induced cytotoxicity and cell proliferation may be critical factors in tumor induction in this tissue.

Effect of Dietary Restriction on DNA-Adduct Formation of Benzo[a]pyrene and 2-Acetylaminofluorene in Mouse Liver

Abstract Dietary restriction (DR) alters hepatic drug metabolizing enzyme activities that affect the metabolic activation of xenobiotics. Previously, we have studied the effect of DR on the formation of benzo[a]pyrene (BaP)-DNA adducts in male Fischer 344 rats. In this study, the effects of DR on the formation of specific BaP-DNA adducts in mouse livers were investigated. In addition, 2-acetylaminofluorene (2-AAF) modified DNA adducts in livers of mice and rats were also examined. DR reduced the body and liver weights of male B6C3F1 mice. Both the total [3H]BaP-DNA binding and the specific BaP-DNA adduct, N2-dG-BaP, detected by 32P-postlabeling technique, were found to be greater in DR mice than in animals fed al libitum (AL). The formation of the 2-AAF-DNA adduct, AF-C8-dG, was not affected by DR. Similar results were obtained from the in vitro DNA adduct formation of these two carcinogens mediated by rat and mouse liver microsomes. The effect of DR on the formation of BaP- and 2-AAF-modified DNA adducts...

Chronic, topical exposure to benzo[a]pyrene induces relatively high steady-state levels of DNA adducts in target tissues and alters kinetics of adduct loss.

Carcinogen-DNA adduct measurements may become useful biomarkers of effective dose and/or early effect. However, validation of this biomarker is required at several levels to ensure that human exposure and response are accurately reflected. Important in this regard is an understanding of the relative biomarker levels in target and nontarget organs and the response of the biomarker under the chronic, low-dose conditions to which humans are exposed. We studied the differences between single and chronic topical application of benzo[a]pyrene (BAP) on the accumulation and removal of BAP-DNA adducts in skin, lung, and liver. Animals were treated with BAP at 10, 25, or 50 nMol topically once or twice per week for as long as 15 weeks. Animals were sacrificed either at 24, 48, or 72 hr after the last dose at 1 and 30 treatments, and after 24 hr for all other treatment groups. Adduct levels increased with increasing dose, but the slope of the dose-response was different in each organ. At low doses, accumulation was linear in skin and lung, but at high doses the adduct levels in the lung increased dramatically at the same time when the levels in the skin reached apparent steady state. In the liver adduct, levels were lower than in target tissues and apparent steady-state adduct levels were reached rapidly, the maxima being independent of dose, suggesting that activating metabolism was saturated in this organ. Removal of adducts from skin, the target organ, was more rapid following single treatment than with chronic exposure. This finding is consistent with earlier data, indicating that some areas of the genome are more resistant to repair. Thus, repeated exposure and repair cycles would be more likely to cause an increase in the proportion of carcinogen-DNA adducts in repair-resistant areas of the genome. These findings indicate that single-dose experiments may underestimate the potential for carcinogenicity for compounds that follow this pattern.

Effect of dietary restriction on partial hepatectomy-induced liver regeneration of aged F344 rats.

Fourteen weeks-old male F344 rats maintained on a reduced caloric diet (60% of ad libitum (AL) food consumption) for 6 weeks or for 14 months did not affect the hepatic cell proliferation in terms of % S phase population, determined by evaluation of DNA synthesis in hepatocytes isolated from either young (5 months) or aged (18 months) rats. However, hepatic basal cellular DNA synthesis estimated by [3H]thymidine incorporation was reduced through acute dietary restriction (DR) in young rats, but increased in aged animals after 14 months restriction. Partial hepatectomy (PH) on aged rats stimulated hepatocyte regeneration and restored some aging-associated biochemical functions, such as drug metabolizing enzyme-dependent xenobiotic metabolic activation which was determined by measuring the formation of carcinogen-DNA adducts. Forty-eight hours after partial hepatectomy, the % of S phase population and the basal nuclear DNA synthesis of hepatocytes isolated from the partial hepatectomized DR-rats were 4- and 2.8-fold, respectively, greater than those of hepatocytes from AL-animals. DR reduced aflatoxin B1 (AFB1) metabolizing enzyme activity and decreased the AFB1-DNA adduct formation in young rats treated with AFB1. In aged AL-rats, the formation of AFB1-DNA adducts diminished to the same level as that of DR-groups and probably was due to the faster decline of drug metabolizing enzymes in aging AL-rats. However, 48 h after PH, the metabolic activation of AFB1 was restored in AL- and DR-groups which resulted in the increase of AFB1-DNA binding by 4.2 and 1.9-fold, respectively. During the liver regeneration of old PH-rats, DR inhibited the AFB1-DNA adduct formation after the PH-rats received a single dose of AFB1. DR increased benzo[a]pyrene (BaP) metabolic activation in both young and aged rats. Aging also decreased BaP-DNA adduct formation in both DR and AL-rats. The increase of BaP-DNA adduct formation in PH-groups was attributed to the restoration of BaP-metabolizing enzyme activity during liver regeneration. The PH-stimulated BaP-DNA adduct formation in AL- and DR-rats was 3.4- and 2.0-fold greater than control aged rats. Our results indicated that the stimulation of PH-induced liver regeneration by DR in aged animals may be attributed to the retardation of aging by DR and the retention of more active biochemical and enzymological functions in old DR-animals.

Cytogenetic and immunofluorescence analysis of benzo[a]pyrene-DNA adduct formation and chromosome damage in larval brain neuroblasts of Drosophila melanogaster.

Recently we have evaluated the relationship between benzo[a]-pyrene(BaP)-DNA adducts, determined by 32P-postlabelling, and clone frequencies in the somatic mutation and recombination test (SMART) in Drosophila melanogaster. Following that study we proceeded to characterise further the mechanism of induction of genetic damage in vivo by BaP in Drosophila by cytogenetic analysis of larval brain neuroblasts. Third stage larvae were treated with 4 and 10 mM BaP for 24, 48 or 72 h. In all cases, the larvae were killed 72 h after the beginning of treatment, entailing 48, 24 or 0 h post-treatment recovery in BaP-free medium, respectively. At the end of the treatment the following data were collected: (i) the types and levels of chromosome aberrations in neuroblast metaphase and anaphase nuclei; (ii) the distribution and level of BaP-DNA adducts, revealed by indirect immunofluorescence in neuroblast nuclei using an anti-(BaP-DNA) antibody. The results indicate that BaP induces chromosome breaks, deletions and exchanges in this system. In particular, chromosome exchanges decrease as the post-treatment recovery time increases, and the dynamics of breaks and deletions appear to be inversely related to those of the exchanges. This suggests that exchanges may require few preconditions to occur and are thus expressed soon after treatment. Chromosome breaks and deletions could require multiple single events before the actual damage is expressed (even some cell divisions away from the end of treatment). The immunofluorescence analysis suggests that BaP-DNA adducts are more abundant in the heterochromatin of the neuroblast nuclei.(ABSTRACT TRUNCATED AT 250 WORDS)

Effects of benzo(a)pyrenf and tetrachlorodibenzo(p) dioxin on fetal dolphin kidney cells: Inhibition of proliferation and initiation of DNA damage

Dolphin kidney cells (CDK) were exposed in vitro to benzo(a)pyrene (BaP) in the presence or absence of 2,3,7,8-tetrachlorodibenzo(p)dioxin (TCDD), a cytochrome P450-inducing agent, and/or α-naphthoflavone (α NF), an inhibitor of cytochrome P450 induction. BaP inhibited mitosis in CDK cells in a dose-dependent manner. TCDD, while inhibiting cell proliferation, did not show a strict dose-dependent mode of action. BaP inhibition of mitosis was decreased by αNF, which also decreased the inhibitory effects of TCDD on CDK proliferation. BaP treatment initiated both 3H-thymidine incorporation and the increased alkali lability of DNA functions of the initiation of excision repair. Cells pre-treated with TCDD and then exposed to BaP exhibited increased BaP-DNA adduct levels and increased DNA excision repair. These data indicate that dolphin cells metabolized BaP in vitro as a function of cytochrome P450-associated activities, that BaP metabolites covalently bound to cellular DNA and initiated excision repair. Inhibition of the cytochrome P450-mediated metabolism of BaP decreased the BaP-associated inhibition of mitosis in dolphin cells.

Effect of Caloric Restriction on the Metabolism of Benzo[a]pyrene (BaP) and the Formation of BaP-DNA Adducts in Male Fischer 344 Rats

Abstract The effect of caloric restriction (CR) on activities of xenobiotic metabolizing enzymes results in alterations in the metabolic activation of chemical carcinogens, with a resultant impact on DNA-carcinogen adduct formation. Using benzo[a]pyrene (BaP) as a model carcinogen, we have studied the effect of CR on the metabolic activation of BaP, in terms of both BaP metabolism and BaP-DNA adduct formation. Male Fischer 344 rats fed CR diets (60% of the food consumption of ad libitum−fed rats) showed higher activities of BaP metabolizing enzymes resulting in increases of BaP metabolism in vitro and BaP-DNA adduct formation in vivo. The results of the study of the effect of CR on the in vitro metabolism of BaP showed that CR increased the total BaP metabolism, as well as BaP-t-9, 10-diol and BaP-t-4, 5-diol formation. However, BaP-t-7, 8-diol, a proximate carcinogenic metabolite of BaP, was decreased by liver microsomes from CR-rats. Our results indicate that the effect of CR on metabolic activation of ...

Relationship between benzo(a)pyrene-DNA adducts and somatic mutation and recombination in Drosophila melanogaster.

The evaluation of the relationship between the dose to DNA of a mutagen/carcinogen and in vivo somatic cell mutagenesis may provide information on the mechanisms leading to induced mutational events. This can be achieved, for example, by coupling test systems that permit the detection of somatic mutation and recombination on the basis of phenotypic changes in cuticular structures of Drosophila melanogaster, with methods for the quantitation of carcinogen-DNA adducts such as the 32P-postlabeling technique. In this article, we evaluate the quantitative relationship between BaP-DNA adduct formation, determined by 32P-postlabeling, and the induction of mutant cells in the wing marker version of the somatic mutation and recombination test (SMART) in Drosophila melanogaster. The total single clones in the trans-heterozygous mwh/flr3 flies show a linear relationship with the BaP-DNA adduct levels, suggesting a single hit mechanism for the genetic damage giving rise to this type of clones. In contrast, the twin clones (which are of recombinational origin) display a linear-quadratic relationship with the adduct levels, suggesting that multiple hits may be involved in generating these clones. The total single clones in the mwh/TM3, Ser flies (in which mitotic recombination is suppressed) show a logarithmic relationship with the adduct levels. The discussion of these data in terms of the pathways that may be involved in the repair of the BaP-DNA adducts leads to the suggestion that in Drosophila melanogaster the repair of Bap metabolite-DNA adducts in somatic cells may proceed, in large part, via post-replicative recombinational repair.