Formation and persistence of benzo[a]pyrene--DNA adducts in different tissues of C57BL/10 and DBA/2 mice

Synchronous fluorescence spectrophotometry (SFS) developed to study benzo[a]pyrene-7,8-diol-9,10-epoxide (BPDE)--DNA adducts was used to measure the formation and disappearance of DNA adducts in the lung, liver, kidney, spleen and small intestine of genetically responsive C57BL/10 (B10) and nonresponsive DBA/2 (D2) mice. After single stomach intubation of 100 mg/kg of benzo[a]pyrene (B[a]P) in both strains, binding of BPDE to DNA reached a peak 48 h after treatment. However, the levels of binding in the lung, liver, kidney and spleen were higher in D2 than in B10 mice. In contrast to this, in the small intestine the higher level of BPDE binding was found in B10 mice and reached its maximum 24 h earlier. Thereafter a very rapid drop in the level of BPDE--DNA adducts to a value of approximately 50% after 48 h was observed in this tissue. In the other tissues of the B10 mice the rate of adducts removal was slower, but by 14 days after treatment 90-100% of adducts were removed. In the D2 mice up to the 4th day after treatment the rates of removal of the BPDE--DNA adducts were similar to that of the B10 mice. Thereafter the level of bound hydrocarbon decreased at a slower rate. During the whole period after B[a]P treatment distinct differences between organs in the amount of BPDE--DNA adducts were observed. In D2 mice the highest level of binding was found in the spleen followed by the lung, kidney, liver and small intestine. In B10 mice the highest level of binding was observed in the DNA of small intestine. The data suggest that the decreased rate of B[a]P metabolism in D2 mice may be at least in some tissues the reason of higher binding of BPDE--DNA adducts in comparison with B10 mice.     

Formation and persistence of novel benzo(a)pyrene adducts in rat lung, liver, and peripheral blood lymphocyte DNA

Male CD rats were injected with single i.p. doses of benzo(a)pyrene (B(a)P), and peripheral blood lymphocytes (PBLs), livers, and lungs were removed at various times after administration. DNA adducts were analyzed in each tissue by 32P postlabeling with nuclease P1 enhancement. Sister chromatid exchange frequencies were concomitantly measured in cultured whole blood. B(a)P-DNA adducts were observed in all three tissues from animals sacrificed between 1 and 56 days after injection. Maximal adduction levels occurred at about 4 days after administration, followed by a gradual loss of adducts over the period examined. The apparent half-lives of total DNA adducts were 15 days in liver, 17 days in PBLs, and 22 days in lung. Induced sister chromatid exchanges were linearly related to the amount of DNA adducts remaining in the PBLs at the time of harvest up to 56 days and were significantly elevated above concurrent controls up to 14 days. One of the major adducts found in each tissue was N2-(10 beta-[7 beta,8 alpha,9 alpha-trihydroxy-7,8,9,10-tetrahydrobenzo(a) pyrene]yl)deoxyguanosine. An additional novel major adduct was found in the liver DNA and is derived from the further metabolism of B(a)P-trans-7,8-dihydrodiol. A second major novel B(a)P adduct was found in the DNA of lung tissues and accounts for about 40% of the total adducts present. Experimental evidence suggests that this adduct is derived from a metabolic pathway that includes the formation of 9-hydroxy-B(a)P.     

Exposure-route-dependent DNA adduct formation by polycyclic aromatic hydrocarbons

Understanding the kinetics of aromatic-DNA adducts in target tissues and white blood cells (WBC) would enhance the applicability of DNA adducts in WBC as surrogate source of DNA in biomonitoring studies. In the present study, rats were acutely exposed to benzo[a]pyrene (B[a]P; 10 mg/kg body wt) via intratracheal (i.t.), dermal and oral administration. DNA adducts were analyzed in relevant target organs and WBC by nuclease P1 enriched (32)P-post-labeling at 1, 2, 4, 11 and 21 days after exposure. Additionally, the internal dose was assessed by measurement of urinary excretion of 3-hydroxy-B[a]P (3-OH-B[a]P). Total B[a]P-DNA adduct levels in WBC were highest after i.t. and oral administration, whereas DNA adducts were hardly detectable after dermal exposure. Highest adduct levels were reached at 2 days after exposure. In lung tissue, DNA adduct levels reached maximal values at 2 days and were highest after i.t., oral and dermal exposure, respectively. DNA adduct levels were significantly lower in WBC as compared with lung. Nonetheless, overall B[a]P-DNA adduct levels in WBC were significantly correlated with those in lung. In target organs, highest DNA adduct levels were observed in skin after topical application, and lowest in stomach after oral administration of B[a]P. Furthermore, DNA adduct levels in WBC were correlated with DNA adduct levels in skin after dermal exposure and stomach after oral administration of B[a]P. Two-fold higher levels of 3-OH-B[a]P were excreted after i.t. administration of B[a]P as compared with dermal or oral exposure. Urinary 3-OH-B[a]P concentrations were correlated with DNA adduct levels at the site of B[a]P application. Overall, it can be concluded that aromatic-DNA adduct levels in WBC can be applied as a surrogate source of DNA for the site of application of B[a]P and reflect binding to lung DNA, independently of the exposure route.     

Mechanism(s) of turmeric-mediated protective effects against benzo(a)pyrene-derived DNA adducts.

The effects of turmeric feeding before and after benzo(a)pyrene [B(a)P] exposure on the levels of B(a)P-derived DNA adducts were studied in tissues of Swiss mice employing (32)P-postlabelling analysis. A reduction in the levels of B(a)P-derived DNA adducts in liver, lung, and forestomach was observed in animals pre-treated with 0.2 or 1% turmeric diet and exposed to B(a)P by oral intubation when compared to animals receiving standard laboratory diet and B(a)P. The observed decrease was not due to dilution caused by nascent DNA synthesis. Comparative evaluation of levels of B(a)P-derived DNA adducts in tissues of animals shifted to 0.2 or 1% turmeric diet after 24 h of oral intubation of B(a)P with those continued on standard laboratory diet did not suggest enhanced disappearance/repair of B(a)P-derived DNA adducts due to exposure to turmeric. Further, pre-treatment of mice with 1% turmeric diet significantly reduced the B(a)P-induced increase in activity of cytochrome P450 (CYP450) isozymes CYP 1A1 and 1A2 in liver, lung, and forestomach of mice. In addition, hepatic glutathione S-transferase (GST) was found to be elevated in turmeric pre-treated mice. Thus turmeric-mediated decrease in induction of phase-I enzymes in liver, lung, and forestomach of mice and enhancement of hepatic GST appear to play an important role in reducing the B(a)P-induced DNA damage in target and non-target tissues.     

Chemoprevention of smoke-related DNA adduct formation in rat lung and heart.

The formation of smoke-related DNA adducts and their chemoprevention were investigated in tissues of male Sprague-Dawley rats, by testing a total of 132 DNA samples by synchronous fluorescence spectrophotometry (SFS), which mainly detects benzo[a]pyrene diolepoxide (BPDE)-DNA adducts. Groups of six animals each were exposed whole-body to mainstream cigarette smoke, once daily, for up to 40 consecutive days. No adduct was revealed in liver DNA, whereas smoke-related DNA adducts were detectable in the lung from the 8th day of exposure and continued to increase until the 40th day. Adducts to heart DNA, which were monitored after 28 and 40 days of exposure, attained even higher levels than those detected in the lungs of the same animals. A high correlation existed between the amounts of smoke-related DNA adducts measured in these two organs. The daily administration by gavage of the thiol N-acetyl-L-cysteine (NAC), an effective mutation and cancer chemopreventive agent, which had been previously shown to inhibit the formation of SFS-positive DNA adducts in rats receiving intratracheal instillations of benzo[a]pyrene, significantly prevented occurrence of the same adducts in both heart and lungs of smoke-exposed rats. No fluorescence signal was observed in liver, lung, or heart DNA of sham-exposed animals. The findings of this molecular dosimetry study complement the results of parallel histopathologic, cytogenetic, biochemical and metabolic analyses of tissues and cells from the same rats, providing evidence for a variety of significant alterations produced by exposure to cigarette smoke and for the specific protective role of NAC.     

Effects of benzyl isothiocyanate and phenethyl isothiocyanate on benzo[a]pyrene metabolism and DNA adduct formation in the A/J mouse

Benzyl isothiocyanate (BITC) inhibits lung tumorigenesis induced in A/J mice by benzo[a]pyrene (B[a]P). In contrast, phenethyl isothiocyanate (PEITC) does not. We tested the hypothesis that BITC inhibits B[a]P tumorigenicity in mouse lung by inhibiting DNA adduct formation, and compared the effects of BITC and PEITC. In mouse liver or lung microsomal incubations, BITC and PEITC inhibited formation of 7,8-dihydro-7,8-dihydroxybenzo[a]pyrene (B[a]P-7, 8-diol) and some other B[a]P metabolites. The metabolism of B[a]P was compared in mouse lung and liver microsomes, 6 or 24h after treatment with BITC or PEITC. In lung, 6 h after treatment, B[a]P-7, 8-diol and some other metabolites were inhibited by BITC and PEITC. However, 24 h after treatment, no inhibition of B[a]P-7,8-diol was observed in microsomes from BITC-treated mice, whereas it was substantially increased in mice treated with PEITC. Effects on B[a]P metabolism in liver microsomes were generally modest. Conversion of B[a]P-7,8-diol to mutagens by mouse liver microsomes was more strongly inhibited by BITC than PEITC. Effects on 7,8-dihydroxy-9, 10-epoxy-7,8,9,10-tetrahydrobenzo[a]pyrene (BPDE)-DNA adduct formation were evaluated in DNA from mice treated with isothiocyanates and B[a]P, and killed 2-120h later. The area under the curve (AUC) for BPDE-DNA adducts in lung was 29.5% less (P = 0. 001) in the BITC-B[a]P treated mice and 19.0% less (P = 0.02) in the PEITC-B[a]P mice than in the mice treated with B[a]P alone. Similar results were obtained in liver DNA. There were no significant differences between the reduction of BPDE-DNA AUC values by BITC versus PEITC. The results of this study support the hypothesis that BITC inhibits B[a]P-induced lung tumorigenesis in A/J mice by inhibiting the metabolic activation of B[a]P to BPDE-DNA adducts. However, differences in BPDE-DNA adduct formation do not appear to explain fully the contrasting effects of BITC and PEITC on B[a]P-induced lung tumorigenesis.     

Effect of tannic acid on rat liver S9 mediated mutagenesis, metabolism and DNA binding of benzo[a]pyrene

Tannic acid, a naturally occurring plant phenol, inhibited rat liver S9 mediated mutagenesis of benzo[a]pyrene in Salmonella typhimurium by 32-77% at concentrations of 5-50 micrograms/mutagenesis plate. Tannic acid (10-40 microM) had no affect on the formation of organosoluble metabolites of benzo[a]pyrene or of its water-soluble conjugates. It did, however, inhibit benzo[a]pyrene (B[a]P) metabolite binding to calf thymus DNA by 40% at a concentration of 40 microM and inhibited benzo[a]pyrene 7,8-dihydrodiol-9,10-epoxide (BPDE): deoxyguanosine adduct formation in calf thymus DNA by 12-54% at concentrations of 10-40 microM. These results suggest that the antimutagenic effect of tannic acid and inhibition of B[a]P metabolite binding to DNA is by a previously described scavenging mechanism and/or by a DNA-affinity binding mechanism that prevents BPDE interaction with DNA as previously described for ellagic acid.     

Fluorescence detection of benzo[a]pyrene--DNA adducts in human lung.

Improved techniques are described for the specific identification of benzo[a]pyrene-diolepoxide (BPDE)--DNA adducts in human tissues. Immunoaffinity chromatography, synchronous fluorescence spectroscopy and second-derivative synchronous fluorescence spectroscopy have previously been used to detect BPDE-DNA adducts in human placenta. Here we report how these methods, together with HPLC and the generation of complete fluorescence excitation--emission matrices, have been used to identify unequivocally BPDE-DNA adducts in samples of human lung. BPDE nucleotide adducts were isolated with immunoaffinity chromatography columns bearing antibodies raised against the (+/-)anti-7,8-diol-9,10-epoxide-deoxyguanosine adduct of benzo[a]pyrene. These adducts were hydrolyzed to tetrahydrotetrols and the hydrolysis products subjected to HPLC. The major product isolated by HPLC, benzo[a]-pyrene-7,10/8,9-tetrahydrotetrol, was determined by fluorescence spectroscopy. Using this method, levels of BPDE-DNA adducts in the range of 1-40 in 10(8) nucleotides were measured in 6 out of 25 samples, with a lower detection limit of one adduct in 10(8) nucleotides. The data may also indicate that adduct levels show regional variation in different parts of the same lung.     

Benzo[a]pyrene-globin adducts detected by synchronous fluorescence spectrophotometry: method development and relation to lung DNA adducts in mice.

A simple synchronous fluorescence spectrophotometry (SFS) to detect benzo[a]pyrene-7,8-diol-9,10-epoxide (BPDE)-globin adducts is described. SFS for BPDE-DNA, which measures detached benzo[a]pyrene (B[a]P)-tetrols after acid hydrolysis of DNA, was applied for BPDE-globin adducts in B[a]P-treated C57BL/6 (B6) mice. Unlike DNA samples, globin is not measurable as such after acid hydrolysis because proteins give a background in SFS. Furthermore, proteinase incubation before acid hydrolysis of globin gave too much background even after purification to be useful in this assay. Of several purification procedures tried after acid hydrolysis (protein precipitation, elution through Sep-Pak C18, filtration, ether extraction of tetrols), the lowest background fluorescence was obtained with ether extractions of B[a] moieties. Ether phases were evaporated to dryness and the remainder dissolved in distilled water (1 ml), which was measured by SFS. Compared to DNA, somewhat milder hydrolysis conditions were optimal for globin samples (0.05 M HCl, 1.5 h, + 90 degrees C). Globin samples from B[a]P-treated mice gave a peak at the same wavelength (345 nm excitation) as the hydrolysis products of BPDE-DNA adducts, indicating B[a]P-tetrols and triols in the sample. Less than half of B[a]P measured in globin was from covalently bound BPDE. In mice injected i.p. with 1-160 mg/kg of B[a]P there was a dose-dependent increase in the amount of BPDE adducts in globin and a positive correlation with lung and liver DNA. Globin adducts were a more sensitive indicator of B[a]P exposure than DNA adducts because more globin can be used for the assay. Although both covalently and non-covalently bound BPDE in globin are detected by SFS, this method is the simplest described so far, reproducible and theoretically sensitive enough for human biomonitoring.     

Relationships between benzo(a)pyrene-DNA adduct levels and genotoxic effects in mammalian cells

The effectiveness of benzo(a)pyrene [B(a)P]-DNA binding as an internal dosimeter was evaluated. Data were obtained from concurrent studies, measuring B(a)P induced genotoxic effects and DNA adducts in several short-term bioassay systems: cytotoxicity, gene mutation, and sister chromatid exchange in Chinese hamster V79 cells; cytotoxicity, gene mutation, and chromosome aberrations in mouse lymphoma L5178Y TK+/-; cytotoxicity and enhanced virus transformation in Syrian hamster embryo cells; and cytotoxicity and morphological transformation in C3H10T1/2CL8 mouse embryo fibroblasts. Both total B(a)P-DNA binding and specific B(a)P-DNA adducts were measured. N2-(10 beta-[7 beta,8 alpha,9 alpha-trihydroxy-7,8,9,10-tetrahydrobenzo(a)pyrene]yl)deoxyguanosine [BPDE I-dGuo] was one of the major adducts identified in all bioassay systems. DNA binding and genotoxic responses varied significantly between bioassays. Each genetic end point was induced with a differing efficiency on a per adduct basis. However, the relationships between frequency of genetic effect or morphological transformation and B(a)P-DNA binding or BPDE I-dGuo were linear within a given assay. In order to compare biological end points of diverse frequencies in diverse biological systems, a doubling adduct level, expressed as the number of BPDE I-dGuo adducts per unit of DNA required to double the induced frequency of biological response, was applied to the data.     

Formation of benzo(a)pyrene/DNA adducts and their relationship to tumor initiation in mouse epidermis

The tumorigenicity of benzo(a)pyrene [B(a)P] applied topically as a skin tumor initiator in Sencar mice and the formation of epidermal B(a)P/deoxyribonucleoside adducts were compared over a similar range of doses (50 to 1600 nmol). The tumor-initiating activity of B(a)P, its covalent binding to mouse epidermal DNA, and the formation of the major hydrocarbon/deoxyribonucleoside adduct showed approximately parallel dose-response curves. The major hydrocarbon/deoxyribonucleoside adduct formed cochromatographed with marker adducts of (N2-(10S-[7R,8S,9R-trihydroxy-7,8,9,10-tetrahydrobenzo(a)pyrene]y) deoxyguanosine while other minor adducts also were observed. The disappearance of DNA-bound products in the epidermis was followed for 21 days after an initiating dose of B(a)P (100 nmol) was applied topically to the mice. The half-lives of the B(a)P/deoxyribonucleoside adducts and the total radioactivity bound to the DNA were 4.5 and 5.5 days, respectively. However, in spite of the loss of measurable DNA-bound material, the tumor yield was unchanged regardless of whether promotion was begun 7 or 21 days after initiation. The results suggest a possible causal relationship between B(a)P/deoxyribonucleoside adduct formation and papilloma formation in mouse skin.     

32P-postlabeling analysis of the formation and persistence of DNA adducts in mammary glands of parous and nulliparous mice treated with benzo[a]pyrene.

The susceptibility of the rodent mammary gland to known chemical carcinogens can vary with the stage of gland development. Full-term pregnancy (parity) can confer permanent structural and functional changes in the gland that are associated with decreased breast cancer risk in humans and protection from mammary carcinogenesis in rodents. In this study, the formation and persistence of benzo[a]pyrene (B[a]P)-derived DNA adducts in vivo was determined in the abdominal mammary organs of adult nulliparous and parous BALB/c mice treated orally with the carcinogen. Mammary DNA isolated from animals in both groups revealed only one major adduct on TLC maps by P1-nuclease 32P-postlabeling analysis. The major adduct co-migrated with the (+)-enantiomer of anti-BPDE-dGp. Much lower levels of the (-)-enantiomer were detected. Most of the adduct quantitated was probably contributed by cells of the stromal compartment, since the gland-free organ (cleared fat pad) generated essentially equivalent profiles and level of adduct by 32P-postlabeling. Comparable levels of the B[a]P-derived adduct were observed in the intact mammary organ of both nulliparous and parous mice during a time course from 1 to 28 days after treatment. In both cases, adduct removal occurred exponentially with a half-life of approximately 16 days. The capacity for de novo formation of reactive metabolites by the mammary organ was demonstrated in vitro: digests of DNA from mammary mince exposed to B[a]P generated an adduct on TLC maps that also co-migrated with the (+)-anti-BPDE-dGp standard. Thus, our cumulative findings confirm the capacity of mammary cells to form potentially carcinogenic DNA adducts; however, the functional changes that occur in the mouse mammary gland as a result of parity did not influence the profile, level or persistence of adduct following exposure to a known carcinogen. Other factors, such as changes in mammary cell cycle kinetics or responsiveness to promotional stimuli may be more causally related to reduction in incidence of neoplasia observed in parous animals.     

Inhibition of benzo[a]pyrene-DNA adduct formation by Aloe barbadensis Miller

The antigenotoxic and chemopreventive effect of Aloe barbadensis Miller (polysaccharide fraction) on benzo[a]pyrene (B[a]P)-DNA adducts was investigated in vitro and in vivo. Aloe showed a time-course and dose- dependent inhibition of [3H]B[a]P-DNA adduct formation in primary rat hepatocytes (1x10(6) cells/ml) treated with [3H]B[a]P (4 nmol/ml). At concentrations of 0.4-250 microg/ml aloe, the binding of [3H]B[a]P metabolites to rat hepatocyte DNA was inhibited by 9.1-47.9%. Also, in rat hepatocytes cultured for 3-48 h with aloe (250 microg/ml) and [3H]B[a]P (4 nmol/ml), [3H]B[a]P-DNA adducts were significantly reduced by 36% compared with [3H]B[a]P alone. Aloe also inhibited cellular uptake of [3H]B[a]P in a dose-dependent manner at a concentration of 0.4-250 microg/ml by 6.3-34.1%. After a single oral administration of B[a]P to male ICR mice (10 mg/mouse), benzo[a]pyrene diol epoxide I (BPDE-I)-DNA adduct formation and persistence for 16 days following daily treatment with aloe (50 mg/mouse) were quantitated by enzyme- linked immunosorbent assay using monoclonal antibody 8E11. In this animal model, BPDE-I-DNA adduct formation was significantly inhibited in various organs (liver, kidney, forestomach and lung) (P < 0.001). When mice were pretreated with aloe for 16 days before B[a]P treatment, inhibition of BPDE-I-DNA adduct formation and persistence was enhanced. Glutathione S-transferase activity was slightly increased in the liver but cytochrome P450 content was not affected by aloe. These results suggest that the inhibitory effect of aloe on BPDE-I-DNA adduct formation might have a chemopreventive effect by inhibition of B[a]P absorption.      

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.     

High DNA damage by benzo[a]pyrene 7,8-diol-9,10-epoxide in bronchial epithelial cells from patients with lung cancer: comparison with lung parenchyma

In the present study, the level of benzo[a]pyrene 7,8-diol-9,10-epoxide-N(2)-deoxyguanosine (BPDE-N(2)-dG) in normal bronchial epithelial cells from non-cancerous bronchus of 22 lung cancer subjects was evaluated and compared to the lung parenchyma. We found very high formation of BPDE-N(2)-dG adduct in samples corresponding to a pure preparation of bronchial epithelial cells with 4-fold interindividual differences in the DNA adduct levels in the range of 36.5-175.4 BPDE-N(2)-dG adducts/10(8) nucleotides in smokers (mean: 84.7+/-38.4; n = 13) and 3-fold differences in the range of 19.7-62.4 in non-smokers (mean: 37.6+/-22.2; n = 3). DNA isolated from the bronchial tissue consisting of bronchial lining epithelium with adjacent lamina propria showed significantly lower BPDE-N(2)-dG formation (P < 0.001) in the range of 0.4-4.2 BPDE-N(2)-dG adducts/10(8) nucleotides (mean: 1.8+/-0.56; n = 6). This difference is clearly related to the procedure used to prepare the bronchial tissue samples leading to the presence of different types of cells. Eight samples from the normal parenchyma did not show measurable adducts, the other 14 samples showed 50-fold variation (mean: 1.7+/-1.5; range 0.1-5.2 adducts/10(8) nucleotides; n = 14). There were considerably higher adduct levels in pure bronchial epithelial cells than in parenchymal tissue (75.8+/-38.8 vs 0.9+/-1.5 adducts/10(8) nucleotides) (P < 0.0002) BPDE-N(2)-dG adduct concentrate almost exclusively in bronchial epithelial cells. The adduct values obtained in bronchial epithelial cells could be considered as 'critical' for the initiation of human lung cancer. The high formation of BPDE-N(2)-dG adducts in bronchial epithelial cells and investigations showing that the profile of mutations induced by BPDE in these cells is similar to that seen in the p53 gene isolated from human lung tumors implicates benzo[a]pyrene as important carcinogen in tobacco-induced lung cancer in human beings.     

Correlation between DNA or protein adducts and benzo[a]pyrene diol epoxide I-triglyceride adduct detected in vitro and in vivo

In this study, we demonstrated the in vitro and in vivo formation of carcinogen-lipid adduct and its correlation with DNA or protein adducts. The lipids from serum or hepatocyte membranes of Sprague-Dawley rats, human serum and standard major lipids were in vitro reacted with benzo[a]pyrene (B[a]P) and B[a]P metabolites. 7, 8-Dihydroxy-9,10-epoxy-7,8,9,10-tetrahydrobenzo[a]pyrene(BPDE-I), an ultimate carcinogenic form of B[a]P, was covalently bound to triglyceride (TG). BPDE-I-TG adducts isolated by thin-layer chromatography (TLC) were further detected by high-performance liquid chromatography. TGs, including triolein, tripalmitin and tristearin, showed positive reactions with BPDE-I. However, cholesterol, phospholipids (phosphatidylcholine, phosphatidyl-ethanolamine, phosphatidyl-inositol and sphingomyelin) and non-esterified fatty acids (palmitic acid, oleic acid, linoleic acid and stearic acid) did not react with BPDE-I. In addition, other B[a]P metabolites (B[a]P-phenols and -diols) did not react with TG. TG appeared to be the most reactive lipid yet studied with respect to its ability to form an adduct with BPDE-I. There was a clear-cut dose-related formation of [1,3-(3)H]BPDE-I-lipid adducts in vitro between TG and [1,3-(3)H]BPDE-I. In an animal study, BPDE-I-TG was also formed in the serum of rats orally treated with B[a]P (25 mg/rat). Also, obvious correlations between [(3)H]B[a]P related-biomolecule adducts (DNA or protein) or lipid damage and the BPDE-I-TG adducts were obtained in various tissues of mice i.p. treated with [(3)H]B[a]P. These data suggest that TG can form an adduct with BPDE-I, as do other macromolecules (DNA, RNA and protein). Therefore, a carcinogen-lipid adduct would be a useful biomarker for chemical carcinogenesis research and cancer risk assessment.     

Oral administration of naturally occurring coumarins leads to altered phase I and II enzyme activities and reduced DNA adduct formation by polycyclic aromatic hydrocarbons in various tissues of SENCAR mice

Several naturally occurring coumarins, to which humans are routinely exposed in the diet, were previously found to inhibit P450-mediated metabolism of benzo[a]pyrene (B[a]P) and 7,12-dimethylbenz[a]anthracene (DMBA) in vitro, block DNA adduct formation in mouse epidermis and inhibit skin tumor initiation by B[a]P and/or DMBA when applied topically to mice. The present study was designed to investigate the effects of two of these compounds, of the linear furanocoumarin type, when given orally (70 mg/kg per os, four successive daily doses), on P450 and glutathione S-transferase (GST) activities and DNA adduct formation by B[a]P and DMBA in various mouse tissues. Imperatorin and isopimpinellin significantly blocked ethoxyresorufin O-deethylase (EROD) and pentoxyresorufin O:-dealkylase (PROD) activities in epidermis at 1 and 24 h after oral dosing. Imperatorin and isopimpinellin modestly inhibited EROD activities in lung and forestomach at 1 h and significantly inhibited PROD activities in lung and forestomach at 1 h after the final oral dose. Twenty-four hours after the final oral dose of imperatorin or isopimpinellin EROD and PROD activities remained inhibited in epidermis and lung. However, forestomach P450 activity had returned to control levels. Interestingly, imperatorin and isopimpinellin treatment inhibited liver EROD activity at 1 h, had no effect on PROD activity at this time point, but elevated both these enzyme activities at 24 h. Elevated EROD and PROD activities coincided with elevated hepatic P450 content. Imperatorin and isopimpinellin treatment also increased liver cytosolic GST activity at both 1 and 24 h after the final oral dose by 1.6-fold compared with corn oil controls. Oral administration of imperatorin and isopimpinellin also had a protective effect against DNA adduct formation by B[a]P and DMBA. Imperatorin pretreatment decreased formation of DNA adducts by DMBA in forestomach. Pretreatment with isopimpinellin led to reduced DNA adduct levels in liver (B[a]P), lung (B[a]P) and mammary epithelial cells (DMBA). These results suggest that imperatorin and isopimpinellin may have potential chemopreventive effects when administered in the diet.     

Detection and identification of benzo[a]pyrene-DNA adducts by [35S]phosphorothioate labeling and HPLC

Two generally applicable [35S]phosphorothioate postlabeling procedures for the HPLC analysis of polycyclic aromatic hydrocarbon (PAH)-DNA adducts have been developed based upon [32P]phosphate postlabeling assays described by Gupta and Randerath et al. In one procedure, benzo[a]pyrene (B[a]P)-modified DNA was digested to nucleoside 3'-phosphates by micrococcal nuclease and spleen phosphodiesterase and the adducted nucleotides were extracted with 1-butanol. The adducted nucleoside-3'-phosphates were 5'-thiophosphorylated by T4 polynucleotide kinase (T4PNK) and adenosine 5'-O-(3-[35S]thiotriphosphate) to yield [35S]B[a]P-nucleoside-5'-phosphorothioate-3'-phosphate adducts. Although thiophosphorylation of B[a]P-DNA adducts was slower than the corresponding phosphorylation reaction, similar recoveries of the postlabeled adducts were achieved with longer incubation times and higher concentrations of T4PNK. A major advantage of this procedure over the 32P-postlabeling procedure is that the resistance of phosphorothioates to degradation by phosphatases allows selective removal of the unlabeled 3'-phosphate from the [35S]B[a]P-nucleoside-5'-phosphorothioate-3'-phosphate adducts by brief treatment with alkaline phosphatase. [35S]B[a]P-nucleoside-5'-phosphorothioate adducts were also prepared using a nuclease P1/prostatic acid phosphatase DNA degradation method. For anti-7,8-dihydroxy-9,10-epoxy-7,8,9,10-tetrahydrobenzo[a]pyrene (BPDE)-modified DNA, overall adduct recoveries were substantially higher with the nuclease P1/prostatic acid phosphatase method (48-51%) than with the micrococcal nuclease/spleen phosphodiesterase/alkaline phosphatase method (22-29%). There were no significant differences in the HPLC profiles of the [35S]phosphorothioate-postlabeled adducts obtained from these two procedures. HPLC analysis of B[a]P-DNA adducts formed in B[a]P-treated hamster embryo cell cultures demonstrated the formation of two major adducts, (+)syn-BPDE-deoxyguanosine-5'-phosphorothioate and (+)anti-BPDE-deoxyguanosine-5'-phosphorothioate, along with other minor adducts. Based upon an overall adduct recovery of 20% and 0.5 mol as the detection limit of this 35S-postlabeling/HPLC assay, the sensitivity of this assay is 1 adduct/10(8) nucleotides for a 60 micrograms DNA sample. This method offers the advantages of using 35S which has a longer half-life and lower radioactive decay energy than 32P and the ability to prepare PAH-DNA adducts at the monophosphorothioate level which greatly facilitates separation of individual 35S-postlabeled PAH-DNA adducts by HPLC.     

Persistence of benzo[a]pyrenediolepoxide DNA adduct in mouse skin

The covalently bound products of [³H] benzo[a]pyrene (BP) were determined in the DNA isolated from the skin of mice 3 weeks after application of the carcinogen. In the relatively repidly proliferating skin the persistence of BP-DNA adducts was observed, 50% of which were unmodified nucleosides. Small amounts of (7R) BPDE I-dG adduct (5% from the initial formation) were found after 3 weeks. The minor adducts observed at 18 h after treatment were excised.