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PubMed1991

Micronuclei in mouse skin cells following in vivo exposure to benzo[a]pyrene, 7,12-dimethylbenz[a]anthracene, chrysene, pyrene and urethane.

Detection of micronuclei (MN) in skin cells from HRA/Skh hairless mice treated with chemical or physical agents may prove informative in qualitative and quantitative studies of skin carcinogenesis. MN induction and cell survival were estimated in cytokinesis-blocked keratinocytes, cultured for 4 days in vitro, after a single topical dose of various organic compounds. Treatment with 2.56 micrograms (10 nmol) 7,12-dimethylbenz[a] anthracene (DMBA) resulted in maximal MN induction in cells removed from skin 12-24 hr after topical administration (79-88 MN/1,000 cells compared with 10-16 MN/1,000 cells in acetone-treated controls). Even in cells removed only 1 hr after DMBA treatment, a significant increase in MN was evident. However, to allow sufficient time for metabolic activation, a sampling time for of 24 hr was adopted for all test substances. Dose-dependent increases in MN were observed with DMBA, benzo[a]pyrene, chrysene, and urethane. Increased numbers of micronucleated cells were detected at the lowest doses administered in the present study (0.128, 0.5, 50, and 50 micrograms, respectively). Although reduced cell recovery occurred following exposure of mice to acetone, pyrene, and other chemicals, there was no evidence that cytotoxicity contributed to MN scored in keratinocytes. Moreover, the probable noncarcinogen, pyrene, failed to induce MN at doses from 2.5 micrograms to 2.5 mg/mouse. These results show that it is possible to assess chemical exposure in skin by measuring cell survival and skin genotoxicity by measuring MN induction in cultured keratinocytes. The available data suggest that MN induction may be a useful indicator of the carcinogenic potential of chemicals applied to the skin.

9,10-Dimethyl-1,2-benzanthracene↗
PubMed1981

Carcinogenicity of the environmental pollutants cyclopenteno-[cd]pyrene and cyclopentano[cd]pyrene in mouse skin.

Cyclopenteno[cd]pyrene (CPEP) is a widespread environmental pollutant. This hydrocarbon and its 3,4-dihydro derivative, cyclopentano[cd]pyrene (CPAP), were tested on skin in a two-stage initiation-promotion experiment in CD-1 mice and by repeated application in Swiss mice. The biological effect of CPEP and CPAP was compared to that of benzo[a]-pyrene (BP). Nine-week-old female CD-1 mice in groups of 30 were treated every other day over a 20-day period at mini-dose levels of 0.18, 0.06 and 0.02 mumol of CPEP or CPAP in acetone. One group was treated with BP at the low mini-dose level. Initiation was followed by twice weekly application of tetradecanoyl phorbol acetate for 40 weeks. In the second experiments, nine-week-old female Swiss mice in groups of 30 were treated at dose levels of 1.8, 0.6 and 0.2 mumol CPEP or CPAP in acetone twice weekly for 30 weeks. One group was treated with BP at the low dose. CPAP was virtually inactive in both studies. In the initiation-promotion experiment CPEP was inactive at the low dose level, whereas BP exhibited significant tumorigenicity. At the medium and high doses CPEP showed weak, but statistically insignificant, tumorigenic activity. Repeated application of CPEP at the high, medium and low doses resulted in tumor incidences of 23, 37 and 57%, respectively. This reverse dose-response may be due to the relatively high cytotoxicity of CPEP, BP, which was compared to CPEP at the low dose, elicited tumors in 100% of the mice. Most of the CPEP-induced neoplasms were malignant and some metastasized to lungs and lymph nodes. The inactivity of CPAP suggests the carcinogenicity of CPEP is probably due to formation of the ultimate metabolite CPEP 3,4-oxide. In view of the abundance of CPEP in environmental and occupational pollutants, its moderately potent carcinogenicity may represent a potential health hazard.

Animals↗
PubMed1985

Effect of ellagic acid and hydroxylated flavonoids on the tumorigenicity of benzo[a]pyrene and (+/-)-7 beta, 8 alpha-dihydroxy-9 alpha, 10 alpha-epoxy-7,8,9,10-tetrahydrobenzo[a]pyrene on mouse skin and in the newborn mouse.

Ellagic acid, quercetin and robinetin were tested for their ability to antagonize the tumor-initiating activity of benzo[a]pyrene (B[a]P) and (+/-)-7 beta, 8 alpha-dihydroxy-9 alpha, 10 alpha-epoxy-7,8,9,10-tetrahydrobenzo[a]pyrene (B[a]P 7,8-diol-9,10-epoxide-2), the ultimate carcinogenic metabolite of benzo[a]-pyrene. Ellagic acid, robinetin or quercetin (2500 nmol) had no tumor-initiating activity on mouse skin, but the topical application of 2500 nmol of ellagic acid 5 min before a tumor-initiating dose of 200 nmol of B[a]P 7,8-diol-9,10-epoxide-2 caused a 59-66% inhibition in the number of skin tumors per mouse that were observed after 15-20 weeks of promotion with 12-O-tetradecanoylphorbol-13-acetate. Similar treatment with 2500 nmol of robinetin or quercetin caused a statistically insignificant 16-24% inhibition in the tumor-initiating activity of 200 nmol of B[a]P 7,8-diol-9,10-epoxide-2 applied 5 min later. Treatment of mice with 2500 nmol of ellagic acid 5 min before the application of 50 nmol of B[a]P inhibited the mean number of skin tumors per mouse by 28-33% after 15-20 weeks of promotion, but these decreases were not statistically significant. Robinetin and quercetin had little or no effect on the tumor-initiating activity of B[a]P on mouse skin. Treatment of preweanling mice with 1/7, 2/7 and 4/7 of the total dose of ellagic acid (300 nmol), robinetin (1400 nmol), myricetin (1400 nmol) or quercetin (1400 nmol) i.p. on their first, eighth and fifteenth day of life, respectively, did not cause the formation of tumors in animals that were killed 9-11 months later. Similar treatment of preweanling mice with the above doses of the phenolic compounds 10 min before the i.p. injection of a total dose of 30 nmol of B[a]P 7,8-diol-9,10-epoxide-2 during the animal's first 15 days of life caused a 44-75% inhibition in the number of diol-epoxide-induced pulmonary tumors per mouse. Similar treatment with these plant phenols had little or no effect on B[a]P-induced pulmonary tumors.

7,8-Dihydro-7,8-dihydroxybenzo(a)pyrene 9,10-oxide↗
PubMed1986

In vivo formation and persistence of DNA adducts in mouse and rat skin exposed to (+/-)-trans-7,8-dihydroxy-7,8-dihydrobenzo[a]pyrene and (+/-)-7 beta,8 alpha-dihydroxy-9 alpha,10 alpha-epoxy-7,8,9,10-tetrahydrobenzo[a]pyrene.

The in vivo DNA adduct formation of (+/-)-trans-7,8-dihydroxy-7,8-dihydrobenzo[a]pyrene (BPD) and (+/-)-7 beta,8 alpha-dihydroxy-9 alpha,10 alpha-epoxy-7,8,9,10-tetrahydrobenzo[a]pyrene (anti-BPDE) were compared and the persistence and disappearance of the adducts in both mouse and rat epidermis determined. BPD (100 nmol/mouse in 150 microliter acetone and 200 nmol/rat in 300 microliter acetone) and anti-BPDE (77 nmol/mouse in 150 microliter tetrahydrofuran and 154 nmol/rat in 300 microliter tetrahydrofuran) were topically applied to 50-day-old male Swiss mice and 35-day-old Wistar rats. To improve the identification of the DNA adducts formed, an acid hydrolysis technique was used to convert the BPD- and anti-BPDE-deoxyribonucleoside adducts formed in mouse and rat skin to BP tetrols. The modified deoxyribonucleosides and BP tetrols obtained by hydrolysis of adducts were isolated by reverse-phase h.p.l.c. At approximately similar doses per unit area of treated skin, the initial total binding of these compounds to epidermal DNA and the level of modified deoxyribonucleosides was approximately 6-fold lower in rat skin epidermis than in mouse skin epidermis. Similar ratios of (+/-)-anti-BPDE-deoxyguanosine (dGuo) to (+/-)-syn-BPDE-dGuo adducts (5.7 and 6.1, determined by h.p.l.c. analysis of BP tetrols obtained by hydrolysis of modified dGuo) were found in both mouse and rat epidermis a short time (6 h) after topical application of (+/-)-trans-BPD. Three hours after topical application of (+/-)-anti-BPDE, the ratios of BP-7,10/8,9-tetrol to 7/8,9,10-tetrol were 9:1 in mouse epidermal DNA and 6:1 in rat epidermal DNA. One and three weeks after application of these two compounds, only (+)-anti-BPDE-dGuo was detected in mouse epidermis; 2 and 0.2% of the initial (+)-anti-BPDE-dGuo level was found to persist in the epidermal DNA from BPD- and anti-BPDE-treated mice respectively. No DNA adducts were detected in rat epidermis 3 weeks after BPD and anti-BPDE treatment. Thus, 3 weeks after topical application of BPD and anti-BPDE to mouse and rat skin, the DNA adducts completely disappeared from rat epidermis while they persisted in mouse epidermis. The results suggest that: the persistence of (+)-anti-BPDE-dGuo may be related to carcinogenesis in mouse epidermis by BPD and anti-BPDE; the complete disappearance of the anti-BPDE-dGuo adduct may also account in part for the relative resistance of tissue from this species to the carcinogenic action of benzo[a]pyrene.

7,8-Dihydro-7,8-dihydroxybenzo(a)pyrene 9,10-oxide↗
PubMed1983

Binding of benzo(a)pyrene and (+/-)-7 beta,8 alpha-dihydroxy-9 alpha, 10 alpha-epoxy-7,8,9, 10-tetrahydrobenzo(a)pyrene to histones.

AKR-2B mouse embryo cells were incubated for 24 hr with [3H]benzo(a)pyrene, and the histones were isolated and analyzed using one- and two-dimensional gel electrophoresis and autoradiography. The results revealed that (a) histones H1, H2A, and H3 incorporated significant amounts of label whereas little or no label was associated with histones H2B and H4 and (b) electrophoresis of the histones in the Triton:acid:urea gel system caused labeled histones to have a slower migration than did the corresponding unlabeled histones. Additional studies such as incubation of (+/-)-7 beta,8 alpha-[3H]dihydroxy-9 alpha,10 alpha-epoxy-7,8,9,10-tetrahydrobenzo(a)pyrene with nuclei resulted in radioactive labeling of histones H1, H2A, H2B, and H3 and of high-mobility-group proteins HMG1 and HMG2. The low levels of label associated with histone H4 in the whole-cell and nuclear studies were further investigated by incubating isolated histones with (+/-)-7 beta,8 alpha-[3H]dihydroxy-9 alpha,10 alpha-epoxy-7,8,9,10-tetrahydrobenzo(a)pyrene. Under these conditions, negligible amounts of radioactivity were associated with H4, while significant labeling of H1, H2A, H2B, and H3 and other nuclear proteins was observed. The results suggest that factors other than the presence of suitable nucleophilic acceptor sites on the histones may be necessary for carcinogen binding.

7,8-Dihydro-7,8-dihydroxybenzo(a)pyrene 9,10-oxide↗
PubMed1995

Differential mutagenicity and cytotoxicity of (+/-)-benzo[a]pyrene-trans-7,8-dihydrodiol and (+/-)-anti-benzo[a]pyrene-trans-7,8-dihydrodiol-9,10-epoxide in genetically engineered human fibroblasts.

DNA repair-deficient (xeroderma pigmentosum group A (XPA)) and DNA repair-proficient (normal) human skin fibroblasts were genetically engineered by transformation with a controllable human cytochrome P450 (CYP)1A1 expression vector. Induction of CYP1A1 enabled these cells to metabolize (+/-)-benzo[a]pyrene-trans-7,8-dihydrodiol (BPD) into a potent cytotoxicant and mutagen. The XPA cells were more susceptible than the normal cells to the cytotoxic effects of both CYP1A1-metabolized BPD and exogenously supplied (+/-)-anti-benzo[a]pyrene-trans-7,8-dihydrodiol-9,10- epoxide (BPDE). Furthermore, the differential cytotoxicity between XPA and normal cells induced by CYP1A1-metabolized BPD was 8.4-fold greater than that induced by exogenously supplied BPDE. The two cell lines had similar CYP1A1 activities, suggesting that a difference in metabolic potential was not the cause of the differential response to BPD. At comparable cytotoxicity in both XPA and normal cells, BPD treatment induced more mutants and more DNA adducts than BPDE treatment did. At similar levels of DNA adducts in XPA cells, the levels of cytotoxicity induced by CYP1A1-metabolized BPD and exogenously supplied BPDE were similar, but CYP1A1-metabolized BPD induced a threefold higher hypoxanthine phosphoribosyltransferase mutation frequency. In contrast, at similar levels of adducts in CYP1A1-expressing normal cells, BPD induced less cytotoxicity and a lower mutation frequency. DNA adducts were identified and quantified by 32P-postlabeling analyses. The principal adduct formed by both CYP1A1-metabolized BPD and exogenously supplied BPDE was 10-beta-(deoxyguanosin-N2-yl)-7 beta,8 alpha,9 alpha-trihydroxy-7,8,9,10- tetrahydrobenzo[a]pyrene, indicating that the differential effects of BPD- and BPDE-induced adducts were not due to a difference in the types of adducts formed. The results of these studies suggest that CYP1A1-metabolized BPD may form adducts preferentially in transcriptionally active genes or that the intracellular concentration of BPDE may influence the balance between cytotoxicity and mutagenicity (or both).

7,8-Dihydro-7,8-dihydroxybenzo(a)pyrene 9,10-oxide↗
PubMed1981

Metabolism of benzo(a)pyrene and benzo(a)pyrene 4,5-oxide in rabbit lung.

For several years our laboratory has been investigating the biotransformation of various environmental pollutants by lung. Studies have been performed with pulmonary subcellular fractions, purified monooxygenase and glutathione transferase enzymes, and preparations having intact cellular structure including the isolated perfused lung and cell fractions enriched in alveolar macrophages, Clara cells and alveolar type II cells. Collectively, these investigations have identified several metabolic factors which may contribute to the pulmonary toxicity mediated by certain polycyclic aromatic hydrocarbons (PAH). First, although lung has low overall cytochrome P-450-dependent monooxygenase activity for many substrates, relative to liver, this activity is localized in only a few cell types and specific activity in certain cell types, such as the non-ciliated bronchiolar epithelial (Clara) cell, can be high. Second, oxidative metabolites of benzo(a)pyrene tend to accumulate in pulmonary tissue due, at least in part, to the low ability of lung (relative to liver) to conjugate and detoxify phenolic, dihydrodiol and epoxide metabolites. Thus, products such as benzo(a)pyrene 7,8-dihydrodiol are available for further cytochrome P-450-dependent oxidation to ultimate carcinogens and cytotoxins. Moreover, the lung is efficient in removing benzo(a)pyrene 4,5-oxide and presumably other oxidized PAH metabolites, from the bloodstream. Consequently, the uptake of relatively stable electrophilic metabolites released by the liver may also contribute to pulmonary toxicity.

Animals↗
PubMed1987

In vitro inhibition of the metabolism and mutagenicity of benzo(a)pyrene and benzo(a)pyrene-7,8-dihydrodiol by naphthazarin and other naphthol derivatives.

Among naphthol derivatives tested in the Ames assay, 5,8-dihydroxy-1,4-naphthoquinone or naphthazarin was found to be the most effective inhibitor of benzo(a)pyrene mutagenicity. The inhibitory activity is due in part to the redox cycling of naphthazarin with the concommitant transfer of reducing equivalents from NADPH to molecular oxygen, thus diverting electrons from cytochrome P-450 enzymes. Metabolite separations showed a decrease in microsomal metabolism of benzo(a)pyrene and of benzo(a)pyrene-7,8-dihydrodoil upon addition of naphthazarin. Since both NADP and dicoumarol inhibited the naphthazarin-stimulated non-stoichiometric consumption of NADPH and oxygen then naphthazarin redox cycling probably involves both DT-diaphorase and NADPH cytochrome P-450 reductase.

Benzo(a)pyrene↗
PubMed1991

Determination of benzo[a]pyrene sulfate conjugates from benzo[a]pyrene-treated cells by continuous-flow fast atom bombardment mass spectrometry.

The level of certain water-soluble hydrocarbon conjugates, such as benzo[a]pyrene sulfates (BP-SO4), is a direct measure of carcinogenic polycyclic aromatic hydrocarbon metabolism and an indication of exposure. A new method, based on continuous-flow high-resolution fast atom bombardment mass spectrometry, has been developed for the analysis of BP-SO4 in the medium of cell cultures treated with benzo[a]pyrene. An organic solvent extract of medium from cultures of the human hepatoma cell line (HepG2) was fractionated by reversed-phase SEP-PAK chromatography and microbore high-performance liquid chromatography (HPLC). The HPLC fraction containing BP-SO4 was collected, dried, and injected into a stream of acetonitrile/water/glycerol that was continuously flowing to the tip of the sample probe which was being bombarded continuously by a beam of high-energy xenon atoms. Molecular anions of BP-SO4 (m/z 347) desorbed from the liquid were analyzed by a high-resolution (m/delta m 5000) mass spectrometer and recorded as a function of time. As little as 1.5 pg of BP-SO4 could be detected with a S/N ratio of 8. The mass spectrometer response was linear with respect to the quantity of BP-SO4 injected over the range from 15 to 625 pg. The results obtained with this method show that the HepG2 cultures metabolized 3% of the benzo[a]pyrene into the BP-SO4 conjugate in 24 h. This procedure, which was used to detect and quantify directly BP-SO4 in culture medium without the use of a radiolabeled precursor, should be generally applicable for analyses of sulfated conjugates resulting from the metabolism of different hydrocarbons.

Benzo(a)pyrene↗
PubMed1991

Metabolism of benzo[a]pyrene and (-)-trans-benzo[a]pyrene-7,8-dihydrodiol by freshly isolated hepatocytes from mirror carp.

The metabolism of benzo[a]pyrene (B[a]P) and (-)-trans-benzo[a]pyrene-7,8-dihydrodiol [(-)-B[a]P-7,8-diol], a major putative proximate carcinogenic metabolite of B[a]P, was compared in freshly isolated hepatocytes from mirror carp, a strain of common carp (Cyprinus carpio, L.). Hepatocytes incubated with 40 microM [3H]B[a]P produced 1.22 nmol equivalents of B[a]P metabolites/mg dry wt of cells/h. Conjugated derivatives represented approximately 65% of all B[a]P metabolites and included glucuronides (38%), glutathione conjugates (21%) and sulfates (6%). About 14% of the total accumulated metabolites of B[a]P determined after 1 h incubations were identified as unconjugated derivatives, predominantly B[a]P-9,10-dihydrodiol and B[a]P-7,8-diol (7.4 and 3.1% of total metabolites respectively), with only traces of B[a]P tetrols (less than 1%). Hepatocytes incubated with 40 microM (-)-[14C]B[a]P-7,8-diol produced 4.78 nmol equivalents of metabolites/mg dry wt during a 1 h incubation, yielding an average rate of metabolism during this time period approximately 53% of that determined after a 5 min incubation. The profile of (-)-B[a]P-7,8-diol metabolites remained constant with incubation time (glucuronides, 30-33%; conjugates with glutathione, 43-46%; polyhydroxylated B[a]P derivatives plus sulfate conjugates, 22-24%). HPLC analysis revealed that polyhydroxylated metabolites amounted to 18% of the total metabolites; thus sulfate conjugates amounted to only 4% of the total metabolites. The trans-2 B[a]P-tetrol, which is the major hydrolysis product of (+)-anti-benzo[a]pyrene-7,8-diol-9,10-epoxide (anti-BPDE), represented approximately 11% of the accumulated metabolites of (-)-B[a]P-7,8-diol. Despite the much larger amounts of BPDE formed from (-)-B[a]P-7,8-diol than from B[a]P, the amounts of B[a]P equivalents covalently bound to cellular DNA were the same following 1 h incubations with either substrate (247 +/- 42 or 212 +/- 42 pmol/mg DNA respectively). Thus biochemical and physiological factors other than the production of BPDE are critically involved in determining the level of DNA adducts in hepatocytes as well as the role of these adducts in hepatocarcinogenesis.

Animals↗
PubMed1982

Structures of covalent adducts derived from the reactions of the 9,10-epoxides of 7,8,9,10-tetrahydrobenzo [a] pyrene and 9,10,11,12-tetrahydrobenzo [e] pyrene with DNA.

The reaction of the racemic mixture of 7 beta, 8 alpha-dihydroxy-9 alpha, 10 alpha-epoxy-7,8,9,10-tetrahydrobenzo [a] pyrene (B[a]PDE) and its enantiomer with DNA is highly stereoselective. About 90% of the adducts are derived from the former enantiomer reacting with the amino group of guanine residues. To investigate this stereoselectively we compared the reactions of 9,10-epoxy-7,8,9,10-tetrahydrobenzo [a] pyrene and 9.10-epoxy-9,10,11-12-tetrahydrobenzo [e] pyrene with DNA. Most of the stereoselectivity seen with B [a] PDE is lost. Both epoxide give mainly adducts on the N2 group of guanine by both cis and trans additions to the epoxide. Other adducts, tentatively identified as deoxyadenosine derivatives, were also detected.

7,8-Dihydro-7,8-dihydroxybenzo(a)pyrene 9,10-oxide↗
PubMed1982

Inactivation of DNA-binding metabolites of benzo[a]pyrene and benzo[a]pyrene-7,8-dihydrodiol by glutathione and glutathione S-transferases.

The binding to DNA of reactive metabolites of trans-7,8-dihydro-7,8-dihydroxybenzo[a]pyrene (BP-7,8-diol) was studied following the incubation of tritiated benzo[a]pyrene (BP) and BP-7,8-diol with nuclei from livers of 3-methylcholanthrene-treated rats. Binding was inhibited to a small extent by glutathione (GSH) alone and to a much greater extent by GSH and cytosol or purified GSH-transferases B and E. In this respect GSH-transferases A and C were also active, but less so. Inhibition of binding of BP-7,8-diol metabolites to DNA mediated by GSH-transferases was associated with the formation of GSH conjugates. The extent of inhibition of binding was similar in incubations of nuclei alone, nuclei and rat liver microsomes, and calf thymus DNA and rat liver microsomes. This indicates that reactive metabolites of BP-7,8-diol, formed either by nuclei or microsomes, are readily accessible to soluble GSH-transferases. GSH and cytosol were also active in inhibiting DNA-binding of reactive metabolites from 9-hydroxybenzo[a]pyrene (9-OH-BP). Thus, in the rat hepatocyte GSH and GSH-transferases may be important in protecting DNA from electrophilic attack by reactive BP-7,8-diol and 9-OH-BP species.

Animals↗
PubMed1984

Metabolic activation of benzo[a]pyrene-7,8-dihydrodiol and benzo[a]pyrene-7,8-dihydrodiol-9,10-epoxide to protein-binding products and the inhibitory effect of glutathione and cysteine.

Trans-7,8-dihydroxy-7,8-dihydrobenzo[a]pyrene (BP-7,8-diol) and the anti-isomer of trans-7,8-dihydroxy-9,10-epoxy-7,8,9,10-tetrahydrobenzo[a]pyrene (BPDE) were found to be activated by microsomes isolated from 3-methylcholanthrene (MC)-treated rats to reactive intermediates that bound covalently to microsomal proteins. The extent of binding was markedly reduced by the presence of reduced glutathione (GSH) or cysteine. Fluorescence spectroscopic studies on the products derived from BP-7,8-diol and BPDE after microsomal activation in presence of GSH or cysteine revealed the formation of a common reactive intermediate with unique fluorescence properties. The involvement of cytochrome P-448 in the activation of BP-7,8-diol and BPDE to protein-binding products was inferred by the requirement for NADPH and almost complete inhibition by alpha-naphthoflavone. Furthermore, microsomes from MC-treated rats could be replaced by a reconstituted system containing purified cytochrome P-448, NADPH-cytochrome reductase and co-factors. The conjugation of the reactive intermediates from BP-7,8-diol and BPDE with GSH or cysteine did not require the presence of either microsomes or cytosol, thus indicating a non-catalytic reaction. These results emphasize the importance of cellular nucleophiles such as GSH and cysteine in the deactivation of reactive benzo[a]pyrene (BP) intermediates and also provides evidence for the further activation of the ultimate carcinogen BPDE to more reactive electrophiles and may thus have relevance concerning the regulation of BP-induced carcinogenesis.

7,8-Dihydro-7,8-dihydroxybenzo(a)pyrene 9,10-oxide↗
PubMed1987

Glutathione depletion suppresses conjugation of benzo[a]pyrene metabolites and (+/-)-trans-7,8-dihydroxy-7,8-dihydrobenzo[a]pyrene metabolites with glutathione but does not affect their binding to DNA in C3H/10T1/2 mouse fibroblasts.

The study was aimed at determining the role of glutathione (GSH) conjugation in the binding of reactive benzo[a]pyrene (BaP) species to DNA of C3H/10T1/2 cells. In order to suppress GSH conjugation cells were depleted of GSH by treatment with buthionine sulfoximine for 18 h and 1-chloro-2,4-dinitrobenzene for 1 h prior to incubation with radiolabelled substrates. Under these conditions GSH levels decreased to less than 1% of the control value. C3H/10T1/2 cells produced GSH conjugates with 7,8-dihydroxy-9,10-oxy-7,8,9,10-tetrahydrobenzo[a]pyrene (BaPDE) comprising 6% of the total metabolites formed from BaP or (+/-)-trans-7,8-dihydroxy-7,8-dihydrobenzo[a]pyrene (BaP-7,8-diol). In GSH-depleted cells formation of GSH conjugates with metabolic products of BaP or BaP-7,8-diol was suppressed to 1% of total metabolites during an 8-h incubation period. Metabolic activation of BaP and BaP-7,8-diol by C3H/10T1/2 cells resulted in the formation of DNA adducts which largely consisted of BaPDE:deoxyguanosine. Depletion of GSH altered neither the degree of DNA binding nor the pattern of DNA adducts to any significant extent. When C3H/10T1/2 cells were co-incubated with microsomes from liver of 3-methylcholanthrene-treated rats for 1 h in order to activate BaP or BaP-7,8-diol extracellularly, the same pattern of GSH conjugates and DNA adducts was generated as by intracellular metabolism of the polycyclic hydrocarbons. No GSH conjugates were detected following co-incubation of microsomes with GSH-depleted C3H/10T1/2 cells. The formation of DNA adducts again remained unaffected by the suppression of conjugation. C3H/10T1/2 cells are apparently capable of conjugating BaPDE with GSH but are not capable of trapping by GSH conjugation those BaPDE moieties which bind to DNA. The results are compatible with the notion that BaPDE is partially contained in a cellular compartment--presumably the lipid environment of membranes--where it is inaccessible to GSH transferases of C3H/10T1/2 cells.

Animals↗
PubMed1987

Human hair follicle benzo[a]pyrene and benzo[a]pyrene 7,8-diol metabolism: effect of exposure to a coal tar-containing shampoo.

Assay systems for the evaluation of carcinogen interaction with human tissues are essential for assessing cancer risk. Hair follicles are a readily available source of human epithelial tissue and offer an excellent system with which to study carcinogen metabolism in human populations. In this study freshly plucked human hair follicles were employed to measure the metabolism of benzo[a]pyrene (BP), benzo[a]pyrene-7,8-diol (BP 7,8-diol), and the enzyme-mediated binding of [3H]-BP to DNA. The effect of human exposure to a crude coal tar (CCT)-containing shampoo, a preparation rich in polycyclic aromatic hydrocarbons (PAHs), on these parameters was also evaluated. Twelve healthy volunteers were studied before and after shampooing their hair daily for 4 days with the CCT-containing shampoo. Wide interindividual variation was observed in basal cytochrome P-450-dependent aryl hydrocarbon hydroxylase (AHH) activity which ranged from 0.6-17.6 fmol water-soluble BP metabolites/h/hair follicle (mean +/- SE of 32 individuals was 9.7 +/- 0.9). After use of the shampoo for 4 days AHH activity increased in 10 of the 12 volunteers (50-148%) and enhancement of enzyme-mediated binding of BP to DNA was detected in most subjects. Hair follicles were shown to convert BP to several metabolic species including BP 7,8-diol, a major precursor of the ultimate carcinogenic metabolite of BP. Benzo[a]pyrene-7,8-diol itself was also metabolized by the human hair follicles in this system. Clotrimazole, a known inhibitor of the metabolism of BP as well as the carcinogenicity of the hydrocarbon in rodent skin, was found to inhibit AHH and the in vitro metabolism of BP and BP 7,8-diol in human hair follicles. Oral administration of a similar antifungal imidazole, ketoconazole at a dose of 200 mg daily for 5 days, to healthy volunteers also resulted in greater than 90% inhibition of hair follicle AHH activity. These studies indicate that hair follicles represent an accessible tissue suitable for assessing the extent of PAH carcinogen metabolism in human subjects. Furthermore, enzyme activity critical to cancer induction by PAHs was shown to be inducible following the use of a CCT-containing shampoo. This carcinogen-activating enzyme system was substantially inhibited by imidazole compounds, suggesting that they may prove effective as anticarcinogens in human populations.

Aryl Hydrocarbon Hydroxylases↗
PubMed1985

Inhibition of benzo(a)pyrene and benzo(a)pyrene-trans-7,8-diol metabolism and DNA binding in mouse lung explants by ellagic acid.

The effect of ellagic acid, a naturally occurring plant phenol, on the binding to DNA and metabolism of benzo(a)pyrene (BP) and trans-7,8-dihydro-7,8-dihydroxybenzo(a)pyrene (BP 7,8-DHD) in cultured explants of strain A mouse lung was investigated. The explants were cultured in a rocking organ culture chamber for 16 h in the presence or absence of 10, 25, 50, and 100 microM ellagic acid. These concentrations of ellagic acid were nontoxic as determined by biochemical and histological methods. The ellagic acid was then removed from the cultures, and the explants were incubated with either 1 microM [3H]BP or [3H]BP 7,8-DHD for 24 h. Explant DNA was isolated using hydroxylapatite chromatography, and the BP metabolites in the medium were analyzed by high-pressure liquid chromatography. Ellagic acid (50 microM) inhibited the binding of BP and BP 7,8-DHD to lung DNA by 46 to 50% and 60 to 70%, respectively. High-pressure liquid chromatography analysis showed that ellagic acid (100 microM) inhibited the metabolism of BP by 20 to 40% and of BP 7,8-DHD by 20%, as indicated by the increased amounts of unmetabolized substrates and decreased amounts of metabolites in the medium. The major BP:DNA adduct in the explants was 7R-N2-[10 beta-[7 beta, 8 beta, 9 alpha-trihydroxy-7,8,9,10-tetrahydrobenzo(a)pyrene]yl: deoxyguanosine, and its formation was reduced by 60 to 65% in the presence of 100 microM ellagic acid. These data suggest that the reduction of BP and BP 7,8-DHD metabolite binding to DNA by ellagic acid may have been due to inhibition of the formation and/or removal of BP 7,8-diol-9,10-epoxide prior to its binding to DNA.

Animals↗
PubMed1977

[Transplacental effect of benz(a)pyrene and pyrene].

The transplacental and direct effect of benzo(a)pyrene (BP) and pyrene on A and C57BL mice and their offspring was studied. BP proved to present blastomogenic danger for the offspring. In A mice offspring the greatest blastomogenic effect was expressed with the dose of 6 mg: lung tumours developed in 76.8% against 12.3% in the control (P less than 0,001). Tumours of the liver were revealed in the offspring of C57BL mice, chiefly in males. Their incidence with the dose of 12 mg of BP was 31.6% in males: and 9.1% in female; in the controls--1.2% in males, in the control females no tumours of the liver were observed. Noncarcinogenic analogue of BP--pyrene produced no blastomogenic effect.

Adenoma↗
PubMed1986

Effect of aliphatic amides on oncogenic transformation, sister chromatid exchanges, and mutations induced by cyclopenta[cd]-pyrene and benzo[a]pyrene.

We examined the effects of the aliphatic amides isopropyl-valeramide (IVA) and allylisopropylacetamide (AIA) on oncogenic transformation and sister chromatid exchanges (SCE) induced by cyclopenta[cd]pyrene (CPP) and benzo[a]pyrene (B[a]P) in C3H/10T1/2 cells and on B[a]Pdiol-epoxide (BPDE)-induced mutation at the HGPRT locus in Chinese hamster ovary (CHO) cells. IVA and AIA significantly suppressed B[a]P and CPP transformation in vitro. Both amides were effective when given just prior to, simultaneously with, or 24 h after carcinogen exposure. On the other hand, IVA and AIA did not affect cytotoxicity, the frequencies of SCE induced by CPP or B[a]P, nor BPDE-induced mutations in CHO cells. These and previous results suggest that the mechanism of inhibition of transformation by IVA or AIA may be very specific and probably not related to the early initiation event in oncogenic transformation in vitro.

7,8-Dihydro-7,8-dihydroxybenzo(a)pyrene 9,10-oxide↗