Polycyclic hydrocarbon activation: bay regions and beyond.
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Animals↗
Biomedical subjects
P L Grover
Publications and source records attributed to P L Grover.
Evidence for the involvement of a bis-diol-epoxide in the metabolic activation of dibenz[a,h]anthracene to DNA-binding species in mouse skin.
Dibenz[a,h]anthracene (DB[a,h]A) and its microsomal metabolites, trans-3,4-dihydro-3,4-dihydroxydibenz[a,h]anthracene (DBA-3,4-diol), trans,trans-3,4:8,9-tetrahydro-3,4:8,9-tetrahydroxydibenz[a,h]anth racene, trans,trans-3,4:10,11-tetrahydro-3,4:10,11-tetrahydroxydibenz[a,h] - anthracene (DBA-3,4,10,11-bis-diol) and trans,trans-3,4:12,13-tetrahydro-3,4:12,13- tetrahydroxydibenz[a,h]anthracene were each applied topically to mouse skin and the epidermal DNA isolated 24 h later. 32P-postlabeling analysis of each of the DNA samples was performed. DNA from mice treated with DB[a,h]A produced an adduct map on TLC consisting of one major and three minor adduct spots. A similar pattern of spots was produced by DBA-3,4-diol. No detectable DNA adducts were produced by trans,trans-3,4:12,13-tetrahydro-3,4:12,13-tetrahydroxy- dibenz[a,h]anthracene, although a single, minor adduct spot was produced by trans,trans-3,4:8,9-tetrahydro-3,4:8,9-tetrahydroxydibenz[a,h]- anthracene. However, DBA-3,4,10,11-bis-diol was found to produce a major single adduct that comigrated on thin layer chromatography with the major adduct produced by both DB[a,h]A and DBA-3,4-diol. In addition, this adduct was present at a level 10 times higher than the corresponding adduct produced by treatment with the parent hydrocarbon. Coelution of the major adducts formed from DB[a,h]A and DBA-3,4-diol with that formed from DBA-3,4,10,11-bis-diol was also demonstrated on reverse-phase high performance liquid chromatography. Thus, we propose that, in mouse skin, the major pathway of DB[a,h]A activation to DNA binding products is via a 3,4-diol to the 3,4,10,11-bis-diol and ultimately to a bis-diol-epoxide (potentially the 3,4,10,11-bis-dihydrodiol-1,2-oxide).
Animals↗
Separation of 32P-labelled nucleoside 3',5'-bisphosphate adducts by HPLC.
Relatively few reported attempts have been made to substitute HPLC for the thin-layer ion-exchange chromatography (TLC) conventionally used in the 32P-postlabelling assay. Using a reverse-phase phenyl-modified silica gel column and a gradient of methanol in 0.5 M sodium phosphate buffer (pH 2.0), we were able to improve the resolution of very similar adducts. Combined with on-line detection of Cerenkov radiation, this method allows separation of sub-femtomole quantities of 32P-labelled nucleoside 3',5'-bisphosphates modified by bulky carcinogens. Using this method, we were able to separate nine of the ten major adducts formed by reaction of the diol-epoxides of ten polycyclic aromatic hydrocarbons with DNA, and resolve different adducts formed by a single carcinogen. The major adducts formed by benzo[b]fluoranthene (BbF) or dibenz[a,h]anthracene in mouse skin in vivo have been shown to be distinct from the adducts formed directly by the bay-region diol-epoxides. The heterocyclic amines IQ and MeIQ have each been shown to form one major DNA adduct in several in vitro and in vivo systems; using HPLC we were able to resolve the two adducts formed by these food mutagens. HPLC is especially useful for the identification of adducts by means of chromatographic comparisons and in the analysis of the multiple adducts formed by complex mixtures of environmental carcinogens. The major adducts formed by benzo[a]pyrene (BaP) and BbF in mouse skin in vivo that were not resolved on TLC were well separated by HPLC and thus a major DNA adduct formed in the skin of mice treated topically with coal tar was found to be derived from BaP rather than BbF.
Animals↗
Summary and discussion of methods and results of the Adult Day Health Care Evaluation Study.
This article summarizes the study results and presents an evaluative summary of the implementation of study methods designed to provide guidance in the degree of confidence with which the results may be accepted and generalized to other situations. Patients who were offered VA-ADHC services in the first phase of this study had significantly higher VA health care costs on average than patients assigned to customary care, with no apparent incremental health benefit to themselves or their care givers. One can have a high level of confidence in these results. The ADHC clinical services were implemented as planned, the randomized controlled trial was implemented successfully, and such threats to validity as insufficient numbers of patients and differential attrition were not present. Certain subgroups of patients assigned to VA-ADHC had VA costs of care that were not significantly higher than those assigned to customary care, although these results must be interpreted with caution. The findings of the second phase of the study evaluating contract ADHC provide no support for choosing to provide either contract ADHC or VA-ADHC over the other. The nonrandomized design and smaller sample size suggest that inferences from the contract ADHC evaluation should be drawn with more caution than those from the VA-ADHC evaluation.
Activities of Daily Living↗
HPLC separation of 32P-postlabelled benzo[b]fluoranthene-DNA adducts.
Analysis using 32P-postlabelling and a recently developed HPLC method resolved the adduct formed by reaction of the benzo[b]fluoranthene (BbF) anti-bay-region diol-epoxide with DNA from the more polar major adduct produced by the hydrocarbon in three different biological systems. In each case, the adduct formed from the anti-bay-region diol-epoxide constituted only a minor proportion of the total DNA modification. Comparisons of the DNA adducts formed from the hydrocarbon with those formed in microsomal incubations from the putative metabolites BbF-9,10-diol, anti-BbF-9,10-diol-11,12-oxide and the 5,9,10- and 6,9,10-BbF-triols indicate that the predominant pathway for BbF activation in skin probably involves a bay-region triol-epoxide possessing a phenolic OH-group on the peninsula ring.
Animals↗
HPLC separation of 32P-postlabelled DNA adducts formed from dibenz[a,h]anthracene in skin.
Mouse skin and human skin have been treated in vivo or in short-term organ culture with dibenz[a,h]anthracene (DB[a,h]A), the related 3,4- or 5,6-diols or the anti- or syn-3,4-diol 1,2-oxides. DNA hydrolysates have been 32P-postlabelled and the adducts present examined by HPLC using a phenyl-modified reverse phase column and, for comparison, by PEI-cellulose TLC and autoradiography. The adducts formed when the diol-epoxides were reacted with salmon sperm DNA were also examined. The results show that in mouse skin treated in vivo, the major adducts formed from DB[a,h]A and the 3,4-diol were the same and that two of them were more polar than those formed in skin or in DNA that had been treated with the related anti- or syn-diol epoxides. Human skin treated with DB[a,h]A in culture yielded an adduct profile that was qualitatively similar to the profiles obtained with mouse skin.
Animals↗
Metabolic activation of the food mutagens 2-amino-3-methylimidazo[4,5-f]quinoline (IQ) and 2-amino-3,4-dimethylimidazo[4,5-f]quinoline (MeIQ) to DNA binding species in human mammary epithelial cells.
When incubated in suspension with the heterocyclic aromatic amine food mutagens 2-amino-3-methylimidazo [4,5-f]-quinoline (IQ) and 2-amino-3,4-dimethylimidazo[4,5-f]quinoline (MeIQ), human mammary epithelial cell aggregates were found, by 32P-postlabelling analysis, to yield DNA that contained adducts. Analysis by HPLC of the 32P-labelled digests of mammary cell DNA indicated that in each case a major adduct peak corresponded to that produced in DNA in vitro by activated derivatives of the two compounds. The patterns of adducts obtained when DNA digests were separated by TLC on polyethyleneimine-cellulose plates were found to resemble those previously shown to be present in DNA of tissues of mice fed IQ or MeIQ. These results demonstrate the ability of human mammary epithelial cells to activate carcinogenic heterocyclic compounds known to be present in the human diet to DNA binding derivatives.
Biotransformation↗
The in vitro metabolic activation of dibenz[a,h]anthracene, catalyzed by by rat liver microsomes and examined by 32P-postlabelling.
DNA has been incubated in vitro with dibenz[a,h]anthracene (DB[a,H]A) and the related 5,6-diol and 3,4-diol in the presence of 3-methylcholanthrene- or Aroclor 1254-induced rat liver microsomes. After incubation, the DNA was extracted and examined for the presence of aromatic adducts using the nuclease P1 modification of the 32P-postlabelling technique. The maps of PEI-cellulose plates and autoradiography showed that 92% of the radioactivity contained in DB[a,h]A-DNA adduct spots is derived from the related 3,4-diol and that about 50% of the adducts may be formed following the conversion of this diol to the bay-region anti- and syn-3,4-diol 1,2-oxides.
Animals↗
Microsomal metabolism of dibenz[a,c]anthracene, dibenz[a,h]anthracene and dibenz[a,j]anthracene to bis-dihydrodiols and polyhydroxylated products.
Polar, ethyl acetate soluble metabolites formed in incubations of dibenz[a,c]anthracene (DB[a,c]A), dibenz[a,h]anthracene (DB[a,h]A) and the related DB[a,h]A 3,4-diol and dibenz[a,j]anthracene (DB[a,j]A) with 3-methylcholanthrene (3-MC)-induced rat liver microsomal preparations have been separated by HPLC and examined using fluorescence, UV and NMR spectroscopy. Metabolites with spectral properties consistant with their identification as the 3,4:8,9-bis-diol of DB[a,j]A and a 1,2,3,4,12,13-hexol derived from DB[a,c]A were found. DB[a,h]A was metabolized to three polar products identified as the 3,4:10,11-bis-diol and the related 1,2,3,4,8,9- and 1,2,3,4,10,11-hexols, which were also formed, together with the related 1,2,3,4-tetrol, from the DB[a,h]A 3,4-diol. The possible role of bis-diols in the metabolic activation of these three dibenzanthracenes is discussed.
Animals↗
The metabolic activation of dibenz[a,h]anthracene in mouse skin examined by 32P-postlabelling: minor contribution of the 3,4-diol 1,2-oxides to DNA binding.
Dibenz[a,h]anthracene (DB[a,h]A) and the related 3,4-diol and anti- and syn-3,4-diol 1,2-oxides were applied to the shaved dorsal skin of groups of four C57Bl/CB1 mice. Twenty-four hours later the mice were killed, DNA isolated from the treated skin, hydrolysed and examined for the presence of aromatic adducts using the nuclease P1 modification of the 32P-postlabelling technique. Autoradiography of the maps obtained by chromatography on polyethyleneimine-cellulose plates showed that six DNA adduct spots that were derived from DB[a,h]A were also present in the DNA of skin treated with the DBA 3,4-diol and that, whilst four of these adduct spots were also seen in maps prepared from the DNA of skin treated with the anti-3,4-diol-1,2-oxide, they were not present in DNA from skin to which the syn-isomer had been applied. The identity of these adduct spots was confirmed by their coincidence when mixtures of different DNA hydrolysates were chromatographed together. Quantitatively, the highest levels of mouse skin modification were obtained with the diol-epoxides and the lowest with DB[a,h]A. The results suggest that most of the DNA adducts formed in DB[a,h]A-treated mouse skin arise through metabolism of the hydrocarbon to the related 3,4-diol and that some may be formed following the conversion of this diol to the bay-region anti-3,4-diol-1,2-oxide.
Animals↗
Evaluation of DNA-binding activity of hydroxyanthraquinones occurring in Rubia tinctorum L.
The naturally-occurring anthraquinones (AQs), alizarin (1,2-dihydroxyanthraquinone) and lucidin (1,3-dihydroxy-2-hydroxymethylanthraquinone), were incubated with DNA in the presence of S9 mix. The isolated DNA was analysed by 32P-postlabelling for the presence of aromatic adducts. Only lucidin formed up to five different DNA adducts in the range from 0.995 to 3.05 adducts/10(8) nucleotides. Lucidin was also incubated with polynucleotides poly[d(A-T)] and polydG*polydC in the presence of S9 mix. Analysis of polydG*polydC revealed a similar adduct pattern to that obtained with lucidin-modified DNA. Alizarin, lucidin, a glycoside mixture containing alizarinprimeveroside and lucidinprimeveroside, and Rubia Teep (a herbal drug made from Rubia tinctorum containing lucidin) were incubated with primary rat hepatocytes for 24 h and the isolated DNA was analysed by 32P-postlabelling. Lucidin, the glycoside mixture and Rubia Teep gave rise to DNA adducts, but alizarin did not. Male Parkes mice were treated orally for 4 days with alizarin (10 mg/d), lucidin (2 mg/d), the glycoside mixture (20 mg/d) or Rubia Teep (1/2 tablet/d) and DNA was isolated from liver, kidney, duodenum and colon. Analysis by 32P-postlabelling revealed that lucidin, the glycoside mixture and Rubia Teep, but not alizarin, formed DNA adducts in all the tissues examined but that the adduct patterns were organ-specific.
Animals↗
Metabolism of 3-hydroxychrysene by rat liver microsomal preparations.
3-Hydroxychrysene, a metabolite of the polycyclic aromatic hydrocarbon (PAH) chrysene, was metabolised by rat liver microsomal preparations obtained from Arochlor 1254-pretreated rats. Eight major metabolites were isolated by high performance liquid chromatography and characterised by u.v. spectroscopy and a variety of mass spectrometric techniques. The metabolites were unambiguously identified as 9-hydroxy-trans-1,2-dihydroxy-1,2-dihydrochrysene and 9-hydroxy-r-1,t-2,t-3,c-4-tetrahydroxy-1,2,3,4-tetrahydrochrysene and tentatively identified as 3-hydroxy-trans-5,6-dihydroxy-5,6-dihydrochrysene (since chrysene is a symmetrical molecule the 3- and 9-positions are equivalent), 9-hydroxy-trans-3,4-dihydroxy-3,4-dihydrochrysene, 1,2,3-trihydroxy-1,2,3,4-tetrahydrochrysene, an oxidised phenol and two diphenols. These results indicate that 3-hydroxychrysene can be further metabolised via a number of different pathways including those involving the formation of phenol- and triol-epoxides.
Animals↗
Effects of 1-ethynylpyrene and related inhibitors of P450 isozymes upon benzo[a]pyrene metabolism by liver microsomes.
The effects of three aryl acetylenes, 1-ethynylpyrene (EP), 2-ethynylnaphthalene (EN) and 3-ethynylperylene (EPE), upon the metabolism of benzo[a]pyrene (BaP) by microsomes isolated from rat liver were investigated. These aryl acetylenes all inhibited the total metabolism of BaP. Formation of BaP 7,8-dihydrodiol and BaP tetrol products by microsomal preparations from rats that had been pretreated with 3-methylcholanthrene (3MC) were preferentially inhibited. The effects of EP upon the metabolism of BaP 7,8-dihydrodiol by microsomes from rat liver were also studied. This aryl acetylene strongly inhibited the formation of BaP tetrols from BaP 7,8-dihydrodiol by liver microsomes both from untreated rats and from rats pretreated with 3MC, but enhanced the conversion of the BaP dihydrodiol into other metabolites.
Animals↗
Formation of DNA adducts in the skin of psoriasis patients, in human skin in organ culture, and in mouse skin and lung following topical application of coal-tar and juniper tar.
Preparations of coal-tar and juniper tar (cade oil) that are used in the treatment of psoriasis are known to contain numerous potentially carcinogenic polycyclic aromatic hydrocarbons (PAH). Evidence of covalent binding to DNA by components of these mixtures was sought in a) human skin biopsy samples from 12 psoriasis patients receiving therapy with these agents, b) human skin explants maintained in organ culture and treated topically with the tars, and c) the skin and lungs of mice treated with repeated doses of the formulations following the regimen used in the clinic. DNA was isolated from the human and mouse tissues and digested enzymically to mononucleotides. 32P-Post-labeling analysis revealed the presence of aromatic DNA adducts in the biopsy samples at levels of up to 0.4 fmol total adducts/microgram DNA. Treatment of human skin in organ culture produced similar levels of adducts, while treatment with dithranol, a non-mutagenic therapeutic agent, resulted in chromatograms indistinguishable from those from untreated controls. In mouse skin, coal-tar ointment and juniper tar gave similar DNA adduct levels, with a similar time-course of removal: maximum levels (0.5 fmol/microgram DNA) at 24 h after the final treatment declined rapidly to 0.05 fmol/microgram at 7 d, thereafter declining slowly over the succeeding 25 d. However, while coal-tar ointment produced only very low levels of adducts in mouse lung (less than 0.03 fmol/microgram DNA), juniper tar produced adducts at a high level (0.7 fmol/microgram DNA) that were persistent in this tissue. These results provide direct evidence for the formation of potentially carcinogenic DNA damage in human and mouse tissue by components of these therapeutic tar preparations.
Administration, Topical↗
Operational efficiency research in Department of Veterans Affairs health services.
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Efficiency↗
Human DNA adducts due to smoking and other exposures to carcinogens.
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Bronchi↗
DNA adduct formation in human and mouse skin by mixtures of polycyclic aromatic hydrocarbons.
32P-Postlabelling analysis has been used to detect the formation in vivo of DNA adducts by components of complex mixtures of polycyclic aromatic hydrocarbons (PAHs) in coal-tar, creosote, bitumen, juniper tar, used engine oils and fuel exhaust condensates. The presence of DNA adducts derived from these agents has been investigated in mouse skin, in human skin explants maintained in short-term organ culture and in human skin in vivo, and the formation of many different PAH-DNA adducts was observed. Similar levels and patterns of adducts were found in DNA from human skin to those in mouse skin, which is known to be susceptible to the carcinogenic activity of PAH mixtures, thus demonstrating the potential hazard to man of epidermal contact with these materials.
Animals↗
Influence of the alkyl substituent on mutagenicity and covalent DNA binding of bay region diol-epoxides of 7-methyl- and 7-ethylbenz(a)anthracene in Salmonella and V79 Chinese hamster cells.
The anti-isomers of the bay region diol-epoxides of the strong carcinogen 7-methylbenz(a)anthracene and of the weak carcinogen 7-ethylbenz(a)anthracene were investigated for mutagenicity in Salmonella typhimurium (reversion of the his - strains TA98 and TA100 to prototrophy) and V79 Chinese hamster cells (acquisition of resistance to 6-thioguanine and ouabain; formation of micronuclei). In addition, in the V79 cells, the levels of the DNA adducts formed were determined by 32P-postlabeling analysis. In terms of mutations per nmol compound administered, the methyl derivative was four to 10 times more potent, depending on the genetic endpoint, than its ethyl congener. However, when the results were expressed as mutations per adduct, the difference between the two diol-epoxides was small. Therefore, a higher level of DNA modification appears to be the major reason for the stronger mutagenicity of the methyl derivative. However, both diol-epoxides had similar half-lives (about 9 min) in physiological buffer, as determined from the decline in mutagenic activity after preincubation of the test compound. These results suggest that the effect of the 7-alkyl group on the extent of reaction with DNA is more a result of steric factors than of a change in the intrinsic chemical reactivity of the diol-epoxides.
Animals↗