Biomedical subjects
Publications and source records attributed to D H Phillips.
The naturally-occurring alkenylbenzene, eugenol, was examined for its ability to form DNA adducts in the livers of mice that had been treated with up to 10 mg of the compound. No adducts were detected by 32P-postlabelling with a limit of detection of 1 adduct in 10(9) nucleotides. Under these conditions adducts were readily detected in liver DNA from the structurally-related hepatocarcinogen safrole.
Engine lubricating oils are known to accumulate carcinogenic polycyclic aromatic hydrocarbons (PAHs) during engine running. Oils from nine petrol-powered and 11 diesel-powered vehicles, in addition to samples of unused oil, were analysed for PAH content and ability to form DNA adducts when applied topically to mouse skin. The levels of 19 PAHs, determined by GC, were in total, approximately 22 times higher in used oils from petrol engines than in oils from diesel engines. Male Parkes mice were treated with 50 microliters of oil daily for 4 days before they were killed and DNA isolated from skin and lung tissue. DNA samples were analysed by nuclease P1-enhanced 32P-postlabelling. Used oils from both diesel and petrol engines showed several adduct spots on PEI-cellulose plates at total adduct levels of up to 0.57 fmol/microgram DNA [approximately 60 times greater than in experiments with samples of unused oil in which adduct levels (0.01-0.02 fmol adducts/microgram DNA) were close to the limit of detection]. Higher adduct levels were generally formed by petrol engine oils than by diesel engine oils. Lung DNA contained similar total adduct levels to those in skin although the adduct maps were less complex. Total adduct levels correlated with extent of oil use in the engine, the total PAH concentration in oils and with the concentrations of certain individual PAHs present in the oils. An adduct spot that co-eluted with that of the major benzo[a]pyrene-DNA adduct accounted for 9-26% of the total adducts in skin DNA, and approximately 8% of the adducts in lung DNA, of mice treated with petrol engine oils. A major, and as yet unidentified, adduct spot comprised up to 30% of the total adducts in skin DNA, and up to 89% of the total adducts in lung DNA, of these animals.
Male and female CDF1 mice were administered a single oral dose of 3 mumol of the food mutagens 2-amino-3-methylimidazo[4,5-f]quinoline (IQ) or 2-amino-3,4-dimethylimidazo[4,5-f]quinoline (MeIQ) and killed 24 h later. DNA was isolated from the livers, lungs, kidneys, colon and forestomach and analysed by 32P-postlabelling for the presence of IQ and MeIQ adducts. Several adduct-enrichment procedures were investigated, including ATP-deficient labelling conditions, butanol extraction and nuclease P1 digestion, and only the ATP-deficient procedure was found to produce the same adduct pattern on polyethyleneimine--cellulose TLC as the standard procedure. Up to nine adduct spots were detected in liver DNA from IQ-treated mice, two of which were not detected in other tissues. The levels of binding in both male and female mice were in the order liver greater than kidney greater than colon greater than forestomach greater than lung. Analysis of DNA from MeIQ-treated mice revealed the presence of up to seven adducts, one of which was detected in liver but not in other tissues. The relative order of DNA binding was kidney greater than liver greater than or equal to colon greater than forestomach greater than lung. As dietary feeding of IQ induces liver, lung and forestomach tumours, and MeIQ induces liver and forestomach tumours in this mouse strain, these binding levels do not correlate with the susceptibility of the organs to carcinogenesis induced by these compounds; the results may indicate the importance of additional factors in determining organ specificity of carcinogenicity.
The effect of environmental pollution on DNA adducts in humans was analysed in a highly industrialized area of Poland. Coded samples of white blood cell DNA were analysed by 32P-postlabelling and immunoassay from three populations: coke workers, exposed occupationally to high levels of polycyclic aromatic hydrocarbons (PAHs); residents of the towns around cokeries (local controls); and residents from rural Poland (countryside controls). Local controls exhibited adduct levels and patterns similar to those of coke workers, while the levels in rural controls were 2-3 times lower. The results, based on coded samples and two different assays, suggest that environmental pollution is likely to contribute to the adduct levels in local controls. Furthermore, the results show that the levels of aromatic adducts in white blood cell DNA do not linearily relate to ambient air levels of PAHs but other sources such as food may be important contributors.
Several well-documented examples of human exposure to carcinogens involve complex mixtures of polycyclic aromatic hydrocarbons (PAHs). Although the biological properties of many pure PAHs have been investigated, less is known about their effects when present as components of mixtures. As the ability to form DNA adducts in vivo is generally indicative of carcinogenic activity of PAHs, we have compared the DNA binding potencies of dibenzo[a,e]pyrene (DB[a,e]P), dibenzo[a,e]pyrene (DB[a,h]P), dibenzo[a,i]pyrene (DB[a,i]P), dibenzo[a,l]pyrene (DB[a,l]P) and benzo[a]pyrene (B[a]P), when applied topically, either singly or in combination, to the skin of male Parkes mice. DNA isolated from the skin and lungs was analysed by 32P-postlabelling. The adducts formed by each PAH exhibited markedly different chromatographic mobilities on polyethyleneimine-cellulose TLC plates. The relative binding potencies of the compounds in both skin and lungs were: DB[a,l]P much greater than B[a]P greater than DB[a,h]P greater than DB[a,i]P greater than DB[a,e]P, in good agreement with their reported carcinogenicities in mouse skin. The majority of adducts were removed from DNA within 21 days of treatment, but low levels of adducts were found to persist for at least 3 months in both tissues. When DB[a,l]P, DB[a,e]P and B[a]P were applied together to mouse skin, a total binding 31% lower than expected was detected, while with a mixture of DB[a,e]P and B[a]P the binding to DNA in skin was 65% higher than expected from the binding levels of the carcinogens when applied singly. Other binary combinations of these three PAHs gave adduct levels similar to the sum of the binding levels of the individual components when applied singly. The results demonstrate the usefulness of 32P-post-labelling for the assessment of the DNA binding potencies of PAHs in mouse tissues, and for the detection of interactions between components of mixtures of carcinogens.
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.
Methylated polycyclic aromatic hydrocarbons are common in the human environment. Many of them are stronger carcinogens than their purely aromatic congeners. They may be metabolized to benzylic alcohols. We report here on biochemical and toxicological characteristics of 1-hydroxymethylpyrene (HMP), a typical representative of this class of compounds. Rat liver cytosol, fortified with 3'-phosphoadenosine-5'-phosphosulfate, converted HMP into its sulfate ester (HMPS), HMPS bound covalently to isolated DNA. In physiological buffer at 37 degrees C, HMPS had a half-life of 2 min, the major decomposition product being HMP. Thus, cyclic activation is possible. When Cl- anions were present at physiological concentrations, an additional reaction product of HMPS, 1-chloromethylpyrene (ClMP), could be identified on the basis of its chromatographic properties and its mass spectrum, using the authentic standard for comparison. ClMP was shorter-lived in buffer than HMPS. ClMP reacted with DNA, the adduct pattern in the 32P-postlabeling analysis being similar, or identical, to that of HMPS. ClMP proved to be a very potent mutagen in Salmonella typhimurium, whereas HMPS, and HMP in the presence of a sulfate-conjugating system, showed strong mutagenicity only when Cl- or Br- ions were present in the exposure buffer. It is concluded that HMPS is capable of reacting with DNA, but is hampered in its distribution by membrane barriers.(ABSTRACT TRUNCATED AT 250 WORDS)
Blood samples were obtained from volunteers who were occupationally exposed to polycyclic aromatic hydrocarbons in a Finnish iron foundry and from referents not known to be occupationally exposed to this class of chemical carcinogens. Aromatic adducts were determined in the deoxyribonucleic acid of white blood cells from the exposed workers with the 32P-postlabeling and immunologic techniques. There was a correlation between the estimated exposure in a particular job and the adduct levels. Jobs of men with high adduct levels (greater than 1 adduct/10(7) nucleotides in the postlabeling assay) included sand preparation, molding, shake-out, and transport. The adduct levels were low in men in pattern making, melting, and fettling. This study suggests that 32P-postlabeling and immunoassay may be useful in monitoring human exposure to known and previously unidentified environmental genotoxic agents.
Using 32P-postlabelling, evidence of DNA adduct formation was sought in six mammalian cell lines, namely Chinese hamster ovary (CHO), human cervical carcinoma (HeLa S3), mouse lymphoma L5178Y tk +/- and L5178Y wild-type, human lymphoblastoid TK6 and Chinese hamster V79, following treatment with benzidine (BZD) in the presence of S-9. Adduct formation was also determined in calf thymus DNA reacted in vitro with N-hydroxy-N'-acetyl-BZD, and in liver DNA from mice given a single intraperitoneal injection of BZD. DNA adducts were detected in the calf thymus DNA sample and in mouse liver DNA, but not in DNA from any of the six cell lines. The absence of adduct formation is consistent with the lack of mutagenicity of BZD in CHO, and V79 and in L5178Y cells at the hprt locus, and in TK6 cells at the tk and hprt loci. These results also suggest that the observed mutagenicity of BZD at the tk locus in L5178Y cells may be due to a mechanism(s) not involving covalent binding to DNA.
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White blood cell DNA adducts were measured in coke workers, local controls and countryside controls using the 32P-postlabelling technique and immunoassay. The methods detected aromatic adducts, including those formed by polycyclic aromatic hydrocarbons. Coke workers are heavily exposed to aromatic compounds, which are also emitted in large amounts into the environment. The two techniques detected a large difference in adduct levels between the coke workers and the countryside controls. The adduct levels in the local controls were substantially higher than those in the countryside controls. As occupational exposure did not account for such a difference, the data suggest that the source of aromatic adducts in local controls is environmental pollution.
Different approaches for measuring occupational exposure to polycyclic aromatic hydrocarbons (PAHs) are presented, (i) determination of PAHs in the workplace by personal sampling, (ii) determination of urinary PAHs and PAH metabolites, and (iii) measurements of aromatic DNA adducts in white blood cells by ultra-sensitive enzyme radioimmunoassay (USERIA) and 32P-postlabelling. Large amounts of PAHs, including benzo[a]pyrene (BP), are released from the coke ovens. Mean PAH exposure levels were reduced by 60% when the workers used masks during work. However, low PAH concentrations were found in the urine of the exposed workers. Approximately 40% of the coke-oven workers had detectable levels of benzo[a]pyrene diol epoxide (BPDE)-DNA adducts in the white blood cells measured by USERIA and 90% had levels of aromatic adducts detectable by the 32P-postlabelling assay. In this study there was a correlation between DNA adduct levels and estimated exposure.
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.
Human lung and bladder DNA has been isolated and purified from either surgical or autopsy specimens. Smoking history details were obtained from patients or their close relatives. Each DNA sample was investigated using the nuclease P1 digestion modification of the 32P-postlabelling procedure. Data are presented for 48 lung and 19 bladder specimens. The samples were subdivided into three groups for data analysis, viz. smokers, former smokers and nonsmokers. The mean adduct levels (adducts per 10(8) nucleotides) in lung samples were: [see text] The chromatographic pattern of bladder DNA adducts for smokers was similar to that for smokers' lung DNA, although less intense. Adduct levels in former smokers tended to be lower than in smokers, although loss of adducts appeared to require several years after cessation of smoking. These findings support a link between DNA adduct levels and cigarette smoking, for both the lung and the bladder. For the former tissue there was a strong linear correlation between adduct levels and the number of cigarettes smoked.
The influence of cytochrome P-450-linked monooxygenase, epoxide hydrolase, UDP-glucuronyltransferase and glutathione S-transferases on the metabolic activation of benzo[a]pyrene (BP) was studied by incubating BP with preparations of rat-liver microsomal and cytosolic fractions in the presence of exogenous DNA. 32P-Postlabelling analysis of the DNA revealed the presence of covalently bound adducts formed by BP, which were visualised as radioactive spots on autoradiographs of thin-layer chromatograms. The effects on the adduct profile of adding different combinations of 1,2-epoxy-3,3,3-trichloropropane (TCPO), UDP-glucoronic acid (UDPGA) and glutathione (GSH) to the incubation mixture were determined. As many as 14 different DNA adducts were resolved and quantitated on the chromatograms, the numbers and quantities of which varied within a large range depending on the incubation conditions. The influence of the enzyme inhibitor and cofactors on the adduct patterns reflects the complex effects of simultaneous enzyme interactions on the metabolic activation of BP.
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.
Analysis by nuclease P1-enhanced 32P-postlabelling assay of DNA isolated from the white blood cells of 53 iron foundry workers was carried out independently in 3 laboratories, and the presence of aromatic DNA adducts was detected. The mean adduct levels in foundry workers varied from 9.2 +/- 23 (laboratory 3) and 12 +/- 10 (laboratory 2) to 26 +/- 43 (laboratory 1) and for the controls from 1.7 +/- 0.7 (laboratory 3) to 3.1 +/- 1.7 (laboratory 1) adducts per 10(8) nucleotides. No effect of smoking was observed in the present study. Each laboratory observed large interindividual variations of adduct levels. Good correlations were found between the results of the 32P-postlabelling assays carried out in the 3 laboratories; the correlation coefficients between laboratories 1 and 2, 1 and 3, and 2 and 3 were 0.61, 0.62, and 0.45, respectively, all being statistically highly significant (p less than 0.01). This interlaboratory comparison of the 32P-postlabelling method indicates the reproducibility of the method and its applicability in occupational exposure monitoring.
Samples of unused or used petrol and diesel engine lubricating oils were applied to the shaved dorsal skin of 4- to 6-week-old male Parkes mice, either as a single treatment (50 microliters/mouse) or as four consecutive daily treatments (50 microliters/application). DNA isolated from the skin 24 h after the final treatment was digested to 3'-mononucleotides and analysed by 32P-postlabelling for the presence of aromatic adducts. Enhancement of sensitivity using butanol extraction or nuclease P1 digestion of the DNA hydrolysates led to the detection of up to eight adduct spots on polyethyleneimine-cellulose thin-layer chromatograms with samples of DNA from skin treated with used engine oils, at levels of 40-150 amol total adducts/micrograms DNA. Multiple treatments with the used oils gave rise to similar patterns of adducts in lung DNA. A single treatment of mouse skin with petrol engine exhaust condensate (50 microliters), or diesel engine exhaust condensate (50 microliters), containing 20 and 46 micrograms benzo[a]pyrene (BaP)/g respectively, gave rise to approximately 75 amol total adducts/micrograms DNA in skin. A significant proportion, 31 and 48% respectively, of the adducts formed by the petrol and diesel engine exhaust condensates co-chromatographed with the major BaP-DNA adduct, but with the used engine oils, only petrol engine oil, and not diesel engine oil, produced significant amounts of an adduct (22% of total) that corresponded to the BaP-DNA adduct.