Monitoring for DNA damage of humans occupationally exposed to methyl bromide.

BACKGROUND: Methyl bromide (MeBr) is a methylating agent, weak mutagen and possible animal carcinogen. A molecular epidemiological study to examine human exposure to, and consequent DNA damage by MeBr was conducted in an area where this agent is used extensively for soil sterilisation in greenhouses. MATERIALS AND METHODS: During the first part of the study, blood samples were collected from 21 persons within 24 hours after use of MeBr for greenhouse sterilisation, as well as from 19 non-exposed subjects. Personal air sampling was also carried out, indicating mean air concentrations for different subjects in the range 11-78 mg/m3. In the second part of the study, an attempt was made to examine professional applicators of MeBr who suffered particularly high exposures (mean exposures, based on personal monitoring 23-165 mg/m3). The levels of N7-methylguanine and O6-methylguanine, two DNA adducts known to be induced by MeBr, were assessed in blood leukocyte DNA. RESULTS: Concerning the first part, two subjects (one exposed and one control) were found to be positive for N7-methylguanine, while none of the blood samples analysed had detectable levels of O6-methylguanine. Among 6 such persons examined during the second part, 2 were found positive for N7-methylguanine while none was positive for O6-methylguanine. CONCLUSION: Within the detection power of this limited study, no significant evidence of induction of DNA damage in blood leukocyte DNA by MeBr was found.

Melanin offers protection against induction of cyclobutane pyrimidine dimers and 6-4 photoproducts by UVB in cultured human melanocytes.

The goal of this investigation was to correlate the melanin content in human pigmentary cells with the generation of UVB-induced photoproducts and to examine the relationship between the melanin content and the removal of the photoproducts. Cultured melanocytes from light-skinned individuals synthesized less melanin and produced more cyclobutane pyrimidine dimers and 6-4 photoproducts upon UVB exposure than did melanocytes from black skin. Tyrosine-stimulated melanogenesis provided protection against DNA damage in both cell types. In another set of pigmented cell lines a ratio between eumelanin and pheomelanin was determined. The assessment of association between DNA damage induction and the quantity and quality of melanin revealed that eumelanin concentration correlated better with DNA protection than pheomelanin. Skin type-I and skin type-VI melanocytes, congenital nevus (CN)-derived cells and skin type-II melanocytes from a multiple-melanoma patient were grown in media with low or high L-tyrosine concentration. The cells were irradiated with 200 J/m2 UVB, and the levels of the photoproducts were determined immediately and after 6 and 24 h. Once again the induction of the photoproducts was mitigated by increased melanogenesis, and it was inversely correlated with the skin type. No significant differences were found for the removal of photoproducts in the cultures of skin types I and VI and CN cells. No indications of a delay in the removal of photoproducts in the melanocytes from the multiple-melanoma patient were found either.

Methyl bromide causes DNA methylation in rats and mice but fails to induce somatic mutations in lambda lacZ transgenic mice.

Following single or multiple oral treatments of rats or lambda lacZ transgenic mice with methyl bromide, methylated DNA adducts (N7- and/or O6-methylguanine) were found at comparable levels in various tissues, including among others the glandular stomach, the forestomach and the liver. Multiple rat treatment resulted in substantial decreases in the repair enzyme O6-alkylguanine-DNA alkyltransferase which were probably due in part to direct interaction of the enzyme with methyl bromide. However, no induction of mutagenesis in the lacZ transgene could be detected in any tissue 14 days after single treatments of up to 50 mg/kg or after multiple treatments of as many as 10 daily treatments of 25 mg/kg MeBr.

Induction of somatic mutations but not methylated DNA adducts in lambdalacZ transgenic mice by dichlorvos.

In order to examine the in vivo genotoxic activity of dichlorvos, lambdalacZ transgenic mice (Muta Mouse) were treated i.p. with single (4.4 or 11 mg/kg) or multiple (5 x 11 mg/kg) doses of this agent and sacrificed 4 h or 14 days post-treatment for DNA adduct measurement or mutant frequency analysis, respectively. Neither methylated DNA adducts nor an increase in mutant frequency were detected in the bone marrow, white blood cells, liver, spleen, lung, brain and sperm cells after the single doses. However, following multiple dosing a statistically significant 3-fold increase in mutant frequency was observed in the liver, while a non-statistically significant increase was observed in the bone marrow. In contrast, dimethylsulphate, a model methylating agent, gave rise to detectable DNA adducts but no increase in mutant frequency following i.p. administration of single (30 mg/kg) or multiple (10 x 6 mg/kg) doses.

DNA adducts, mutant frequencies and mutation spectra in lambda lacZ transgenic mice treated with N-nitrosodimethylamine.

Groups of lambda lacZ transgenic mice were treated i.p. with N-nitrosodimethylamine (NDMA) as single doses of 5 mg/kg or 10 mg/kg or as 10 daily doses of 1 mg/kg and changes in DNA N7- or O6-methylguanine or the repair enzyme O6-alkylguanine-DNA alkyltransferase (AGT) were followed for up to 14 days in various tissues. Adduct induction in the liver exceeded by at least one order of magnitude than observed in the next nearest target tissue (lung), and was approximately linearly related to dose, except for O6-methylguanine after the first dose of 1 mg/kg which was lower than expected. Substantial induction of lambda lacZ mutagenesis was observed only in the liver, where the mutant frequency was already maximal within 7 days after 5 mg/kg NDMA and remained unchanged thereafter up to 49 days. Small but marginally significant increases in mutant frequency were consistently observed in the spleen after all three modes of treatment. A lack of proportionality between mutation induction and the administered dose or the corresponding adduct levels was observed, probably reflecting the importance of toxicity-related cell proliferation caused by NDMA at higher doses. Twenty eight days after a dose of 10 mg/kg (causing a 3.6-fold increase in mutant frequency), NDMA was found to increase the frequency of GC-->AT mutations (with a concomitant shift of their preferential location from CpG sites to GpG sites), which made up approximately 60% of the induced mutations. Surprisingly, NDMA also caused a significant increase in deletions of a few (up to 11) base-pairs (22%).

Monitoring of occupational exposure to polycyclic aromatic hydrocarbons in a carbon-electrode manufacturing plant.

An investigation is presented of occupational exposure to polycyclic aromatic hydrocarbons (PAH) in a carbon-electrode manufacturing plant, as assessed by three monitoring methods, viz. environmental monitoring of the external dose by analysis of personal air samples, biological monitoring of the internal dose by analysis of urinary 1-hydroxypyrene (1-OHpyrene), and biological effect monitoring by dosimetry of PAH-DNA adducts in blood lymphocytes. On the basis of job conditions, workers at the plant were divided into three groups with presumed low, intermediate and high exposure to air-borne PAH, respectively. All air samples showed levels of total PAH below the current MAC-value in the Netherlands, which is 200 micrograms/m3, whereas the benzo[a]pyrene level was occasionally higher than the recommended concentration of 2 micrograms/m3. The values of 1-OHpyrene in urine from the intermediate and high exposure groups were significantly higher than those of the low exposure group, namely 3.6- and 8.2-fold, respectively. Clear external and internal exposure was thus demonstrated for workers of the high and intermediate exposure groups, but this did not result in a measurable effect at the DNA level in blood lymphocytes. Tobacco smoking, on the other hand, caused a significant increase of the levels of PAH-DNA adducts but did not affect 1-OHpyrene values. These data suggest that smoking is a more important risk factor for adverse health effects, i.e. cancer, than occupational exposure to PAH in this plant.

DNA damage and mutagenesis induced by procarbazine in lambda lacZ transgenic mice: evidence that bone marrow mutations do not arise primarily through miscoding by O6-methylguanine.

The DNA damaging and mutagenic activities of procarbazine, a methylating drug employed in cancer chemotherapy and suspected of causing therapy-related leukaemia, were investigated in the liver and bone marrow of lambda lacZ transgenic mice (MutaMouse). The drug was administered using two different protocols, a 'high-dose' one involving 5 daily doses of 200 mg/kg, expected to cause depletion of the repair enzyme O6-alkylguanine-DNA alkyltransferase (AGT) and thus favour the selective accumulation of the premutagenic lesion O6-methylguanine (O6-meG) relative to other adducts, and a 'low-dose' one involving 10 daily doses of 20 mg/kg procarbazine. Substantial accumulation of O6-meG was observed in both tissues examined 6 h after the end of the 'high-dose' treatment, with the liver accumulating somewhat higher levels than the bone marrow (28.0 +/- 1.8 fmol/microg DNA and 18.5 +/- 1.1 fmol/microg DNA respectively). However, significant increases in mutant frequency 10 days after the end of treatment were observed only in the bone marrow, reaching a 16-fold increase over background following the 5 x 200 mg/kg treatment. Sequence analysis of the mutations induced after this treatment revealed a mixed spectrum, in which G:C-->A:T transitions (characteristic of O6-meG miscoding) were only a secondary feature: Among 20 mutants analysed, only six such mutations were found, including three at CpG sites, which might have arisen from deamination of 5-methylcytosine. The other mutations observed included 1 A:T-->G:C transition, five transversions (one G:C-->T:A, one double G:C-->C:G, two A:T-->T:A, one A:T-->C:G), five deletions and three insertions. The mechanistic and clinical significance of these findings is discussed.

The use of benzo[a]pyrene diolepoxide-modified DNA standards for adduct quantification in 32P-postlabelling to assess exposure to polycyclic aromatic hydrocarbons: application in a biomonitoring study.

The 32P-postlabelling assay is one of the most sensitive methods for detection of DNA adducts induced by exposure to genotoxic chemicals. Under optimal conditions, detection limits of one adduct per 10(9)-10(10) nucleotides have been reported. This sensitivity now allows monitoring of occupational and even environmental exposure of humans to certain classes of chemicals, mainly polycyclic aromatic hydrocarbons (PAH). Despite its widespread use, 3P-postlabelling is still not a standardized method. Rigorous interlaboratory comparisons are scarce, and those that have been undertaken often show rather different results, both in relative and in absolute values, for the amounts of DNA adducts in the same samples. Furthermore, the optimization of many steps in the procedure has still not been given adequate attention. This paper deals with some technical aspects of detection of PAH-DNA adducts by 32P-postlabelling, in particular with assay calibration and adduct quantification. For this purpose, benzo[a]pyrene (BP)-modified DNA standards were prepared, the adduct contents of which were determined by use of an independent fluorometric method, viz. synchronous fluorescence spectrophotometry (SFS). These BP-DNA standards are processed along with the test samples throughout the entire 32P-postlabelling procedure, from the enzymic digestion up to and including the determination of radioactivity in adduct spots on the chromatogram. As such, these reference samples can be considered as external standards for inter-assay calibration. This method for adduct quantification was compared with the commonly used relative adduct labelling (RAL) and comparative dAMP labelling, which appeared to give rise to an underestimation of adduct levels. The method was applied in a biomonitoring study among workers in a carbon-electrode manufacturing plant, exposed to PAH. Although DNA adduct levels in peripheral blood lymphocytes of exposed workers, as determined by 32P-postlabelling, were not significantly different from those of controls, a significant difference was seen when smokers and non-smokers were compared.

Determination of N7- and O6-methylguanine in rat liver DNA after oral exposure to hydrazine by use of immunochemical and electrochemical detection methods.

Hydrazine belongs to a group of compounds for which there is evidence that the in vivo genotoxic effects become manifest only upon exposure to toxic dose levels. The present study was performed to investigate whether this phenomenon is also reflected in the pattern of DNA methylation. The induction of N7- and O6-methylguanine (MeGua) was studied in liver DNA of rats, 16 hr after treatment with various doses of hydrazine. After DNA isolation, the presence of N7-MeGua in DNA was assessed with an immunochemical method and with a physicochemical technique (HPLC with electrochemical detection). Application of these two methods resulted in almost identical patterns of dose-dependent induction of guanine N7-methylation in rats dosed orally with 0.1 to 10 mg hydrazine per kilogram of body weight, increasing from 1.1-1.3 to 39-45 N7-MeGua per 10(6) nucleotides. At lower dosages a constant adduct level was observed, equivalent to that in untreated rats (background level). The O6-MeGua level was analyzed by a combination of HPLC separation and competitive radioimmunoassay. A background level was observed for untreated rats and no increase was visible up to the 0.2 mg/kg dose group. After hydrazine doses from 0.2 to 10 mg/kg, O6-MeGua increased from 0.29 to 134 per 10(9) nucleotides. These data show that even at dosages below the maximum tolerated dose (0.6 mg/kg/day), for which carcinogenic effects have not been described, DNA adducts are formed. A comparison is made of the data obtained in this study with models that describe the mechanism of hydrazine-induced DNA methylation.

UVB-induced mutagenesis in hairless lambda lacZ-transgenic mice.

UVB-induced mutagenesis was studied in hairless 40.6 transgenic mice (MutaMouse), which contain the lambda gt10lacZ shuttle vector as a target for mutagenesis. Mice were exposed at the dorsal side to either single doses of 200, 500, 800, or 1000 J/m2 UVB or to two successive irradiations of either 200 and 800 J/m2 UVB, with intervals of 1, 3, or 5 days, or to 800 and 200 J/m2 UVB with a 5-day interval. At 23 days after the last exposure, lacZ mutant frequencies (MF) were determined in the epidermis. The lacZ MF increased linearly with increasing dose of UVB. The mutagenic effect of two successive irradiations appeared to be additive. The UV-induced mutation spectrum was dominated by G:C --> A:T transitions at dipyrimidine sites. DNA-sequence analysis of spontaneously mutated phages showed a diverse spectrum consisting of insertions, deletions and G:C --> A:T transitions at CpG sites. The results indicate that the hairless lambda lacZ-transgenic mouse is a suitable in vivo model for studying UVB-induced mutations.

Biomonitoring human exposure to environmental carcinogenic chemicals.

A coordinated study was carried out on the development, evaluation and application of biomonitoring procedures for populations exposed to environmental genotoxic pollutants. The procedures used involved both direct measurement of DNA or protein damage (adducts) and assessment of second biological effects (mutation and cytogenetic damage). Adduct detection at the level of DNA or protein (haemoglobin) was carried out by 32P-postlabelling, immunochemical, HPLC or mass spectrometric methods. Urinary excretion products resulting from DNA damage were also estimated (immunochemical assay, mass spectrometry). The measurement of adducts was focused on those from genotoxicants that result from petrochemical combustion or processing, e.g. low-molecular-weight alkylating agents, PAHs and compounds that cause oxidative DNA damage. Cytogenetic analysis of lymphocytes was undertaken (micronuclei, chromosome aberrations and sister chromatid exchanges) and mutation frequency was estimated at a number of loci including the hprt gene and genes involving in cancer development. Blood and urine samples from individuals exposed to urban pollution were collected. Populations exposed through occupational or medical sources to larger amounts of some of the genotoxic compounds present in the environmental samples were used as positive controls for the environmentally exposed population. Samples from rural areas were used as negative controls. The project has led to new, more sensitive and more selective approaches for detecting carcinogen-induced damage to DNA and proteins, and subsequent biological effects. These methods were validated with the occupational exposures, which showed evidence of DNA and/or protein and/or chromosome damage in workers in a coke oven plant, garage workers exposed to diesel exhaust and workers exposed to ethylene oxide in a sterilization plant. Dose reponse and adduct repair were studied for methylated adducts in patients treated with methylating cytostatic drugs. The biomonitoring methods have also demonstrated their potential for detecting environmental exposure to genotoxic compounds in nine groups of non-smoking individuals, 32P-postlabelling of DNA adducts being shown to have the greatest sensitivity.

Effect of eugenol on the mutagenicity of benzo[a]pyrene and the formation of benzo[a]pyrene-DNA adducts in the lambda-lacZ-transgenic mouse.

To study the possible reduction by eugenol of the mutagenicity and genotoxicity of benzo[a]pyrene (B[a]P) in vivo, the lambda-lacZ-transgenic mouse strain 40.6 (Muta Mouse) was used. Male mice were fed a diet containing 0.4% (w/w) eugenol or a control diet for 58 days. On day 10, half of the mice received an i.p. dose of 100 mg/kg b.w. B[a]P. The lacZ mutants were recovered by packaging of DNA isolated from liver into lambda phage, and expressed in E. coli C lacZ-recA-galE- bacteria. In both control mice and mice fed the eugenol diet, B[a]P treatment resulted in a similar, significant increase in lacZ mutant frequency. Eugenol was not mutagenic by itself. By 32P-postlabelling analysis of the liver DNA using an analysis method with chromatographic conditions for B[a]P-DNA adducts, no effect of eugenol on the formation of B[a]P-DNA adducts in the lambda-lacZ-transgenic mouse was found. By 32P-postlabelling analysis using an alkenylbenzene solvent system the amount of B[a]P-DNA adducts was lower in mice fed the eugenol diet than in mice fed the control diet but the decrease was not statistically significant. However, one spot indicative of an eugenol-associated DNA adduct was detected. The present data provide no evidence for antimutagenic or antigenotoxic potential of eugenol in vivo. Furthermore, they suggest genotoxicity in vivo of eugenol per se.

Comparison of the X-gal- and P-gal-based systems for screening of mutant lambda lacZ phages originating from the transgenic mouse strain 40.6.

The recent introduction of the phenyl-beta-D-galactopyranoside (P-gal)-based positive-selection system for screening of lambda lacZ phages originating from the lambda lacZ transgenic mouse (Muta Mouse) has made the determination of mutant frequencies (MF) a much simpler task. Previously, MF data from these mice have been collected by means of the 5-bromo-4-chloro-3-indolyl-beta-D-galactopyranoside (X-gal) colour-screening procedure. To determine whether data obtained with the two systems are comparable, the MF in lambda phages recovered from liver and brain of transgenic mice treated with N-ethyl-N-nitrosourea (ENU) and liver of benzo(a)pyrene (B(a)P)-treated mice was determined with both procedures. For the livers of mice treated with ENU, both methods yielded approximately the same MF values. No induction of mutants, relative to the control animals, was seen after 1.5 h, but a clear 4-fold increase was measured with both assays at the 14-day time point. No induction of mutants was found in the brain with either method. In the B(a)P-treated mice, both methods showed a substantial induction in MF after 21, 28 and 35 days. The values generated by the X-gal and P-gal methods were not significantly different, with the exception of the 35-day post-treatment point that appeared higher in the X-gal assay. When the mutants isolated by use of the X-gal method were tested in the P-gal assay, a number of these did not turn up as mutants, and the significance disappeared. In conclusion, the data obtained with the two screening procedures agree to such an extent as to permit a direct comparison between the earlier results generated with X-gal and P-gal values generated with the new positive-selection method. This is likely to apply also to other organs and mutagens than those studied here.

Molecular dosimetry of DNA damage induced by polycyclic aromatic hydrocarbons; relevance for exposure monitoring and risk assessment.

Polycyclic aromatic hydrocarbons (PAH) form a large group of organic chemicals that are widely distributed in our environment as pollutants of air, water and soil. Several PAH are carcinogenic in rodents, while exposure to these compounds has been associated with various types of human cancer. Upon entering the body, PAH may be converted into reactive electrophilic species, which can give rise to the formation of DNA adducts. DNA adduct formation is considered to be the initial event in chemical carcinogenesis. In this paper, two methods are illustrated that are widely used to determine PAH-DNA adduct formation, namely 32P-postlabelling, and immunochemical analysis with specific antibodies. The applications of the 32P-postlabelling assay comprise the following: A study of interspecies differences in PAH bioactivation in vitro, with microsomal preparations isolated from liver tissue of various rodent species and of human origin; the results indicate that there are considerable qualitative differences between the adduct patterns obtained, which is relevant with respect to extrapolation from animal to man. The analysis of DNA adduct formation in fish retrieved from marine environments polluted to various extents with PAH; results of these studies show a correlation between liver-DNA adduct levels in these fish and the degree of PAH contamination in the aquatic environment. Biomonitoring of PAH exposure through analysis of adducts in blood cells obtained from heavy and light smokers; the data show a fair correlation between PAH-DNA adduct levels in white blood cells and cotinine content in blood plasma, the latter being used as a marker for exposure to cigarette smoke.(ABSTRACT TRUNCATED AT 250 WORDS)

Role of the intestinal microflora in the formation of DNA and haemoglobin adducts in rats treated with 2-nitrofluorene and 2-aminofluorene by gavage.

The role of the intestinal microflora in the metabolic activation of nitroarenes and arylamines was studied in female Wistar rats that received a dose of 1 mmol/kg 2-aminofluorene (2-AF) in sunflower oil by gavage. Another group received the same dose of 2-nitrofluorene (2-NF). A third group of animals was used as controls. Germfree (GF) rats, GF rats with a rat microflora (RM) and GF rats with a human microflora (HM) were treated. After treatment with 2-AF significant differences were observed in the formation of haemoglobin (Hb) adducts and DNA adducts. The 2-AF-Hb adduct level (mean +/- SD) observed in GF rats (0.57 +/- 0.13 mumol/g Hb) was considerably lower than that observed in RM rats (5.1 +/- 0.6) and in HM rats (6.2 +/- 1.3). DNA adduct levels showed the opposite pattern: levels of adducts co-migrating with deoxyguanosin-8-yl-aminofluorene (dG-C8-AF) in liver tissue were higher in GF rats (4.6 +/- 1.4 fmol/micrograms DNA) as compared to RM rats (2.6 +/- 0.04) or HM rats (2.0 +/- 0.7). In lung tissue and white blood cells a similar influence of the intestinal microflora on DNA adduct levels was observed. These results suggest that the intestinal microflora cleaves conjugates of 2-AF or N-hydroxy-2-AF, thus facilitating enterohepatic recirculation of these compounds and enhancing the formation of reactive intermediates binding to Hb. The latter is not observed for DNA adduct formation, indicating that most of these adducts have been formed after a single passage through the liver. After treatment with 2-NF, Hb and DNA adduct levels were much lower. An adduct spot was observed that was not present in rats that received 2-AF. In GF animals only very low levels of DNA adducts co-migrating with dG-C8-AF or deoxyguanosin-8-yl-acetyl-aminofluorene and no Hb adducts were observed, indicating that the metabolic activity of the microflora is an essential step in both Hb and DNA adduct formation.

Formation and repair of benzo[a]pyrene-DNA adducts in cultured hamster tracheal epithelium determined by 32P-postlabeling analysis and unscheduled DNA synthesis.

Hamster tracheal organ cultures were used to investigate the relationship between DNA adduct formation measured directly by the 32P-postlabeling assay, and the DNA damage measured indirectly by the unscheduled DNA synthesis (UDS) assay. Hamster tracheas were treated with three concentrations of benzo[a]pyrene (B[a]P) for 2 days. Postlabeling and UDS assays were also carried out a few days after removal of the B[a]P. Furthermore, the types of B[a]P-DNA adducts formed in the in vitro organ culture were qualitatively compared with those formed in vivo after intratracheal intubation of B[a]P attached to Fe2O3 particles. In vivo only one adduct was detected by 32P-postlabeling. This adduct cochromatographed with the trans-addition produce of dG and (+)-anti-7,8-dihydroxy-9,10-epoxy-7,8,9,10-tetrahydrobenzo[a]pyrene (BPDE). In vitro, a clear B[a]P-DNA adduct pattern was also found with the 32P-postlabeling assay. Four different adducts were found. The main adduct spot migrated to the same position on the thin-layer chromatogram as the in vivo adduct. B[a]P-DNA adduct formation was both time- and dose-dependent. During the first day after removal of B[a]P the adduct levels still increased, thereafter they decreased at all B[a]P concentrations. A time- and dose-dependent increase in UDS was observed in the tracheal epithelial cells treated with B[a]P in vitro. After removal of the B[a]P, UDS decreased immediately, in contrast to the formation of DNA adducts. The results of the present study show that B[a]P induces time- and dose-dependently both DNA adducts and UDS in hamster tracheal organ culture. Moreover, the main DNA adduct formed in vitro, dG-(+)-anti-BPDE, was the same as that found in vivo.

Improvements in the 32P-postlabelling procedure to quantify bulky aromatic DNA adducts.

This contribution describes methodological modifications and improvements that may contribute to inter-assay reproducibility and more accurate adduct quantification for 32P-postlabelling. Firstly, an anion-exchange chromatography procedure was developed to determine the amount of DNA used per assay and to check its purity, in particular to verify the absence of contaminating RNA. Secondly, calibration standards were prepared, in order to correct for differences in recovery. The modification levels of these standards were determined by synchronous fluorescence spectrophotometric analysis. Thirdly, the effect on adduct levels of exposure to light during postlabelling was investigated. Exposure of polyaromatic DNA adducts on a PEI-cellulose plate reduced the amounts of adducts detected considerably.

Detection of benzo[a]pyrene-DNA adducts in cultured cells treated with benzo[a]pyrene diol-epoxide by quantitative immunofluorescence microscopy and 32P-postlabelling; immunofluorescence analysis of benzo[a]pyrene-DNA adducts in bronchial cells from smoking individuals.

Monoclonal antibodies were raised against the reaction product of benzo[a]pyrene diol-epoxide (BPDE) and deoxyguanosine-5'-monophosphate. The antibodies were used for detection of DNA adducts in situ in BPDE-treated cultured human fibroblasts by immunofluorescence microscopy. Analogue-digital conversion of the fluorescence signal and further image processing allowed measurement of the immunospecific fluorescence in the nuclei of these cells. The results are compared with the adduct levels measured in isolated DNA by 32P-postlabelling. Preliminary results are shown of the application of the immunofluorescence method to the analysis of DNA adducts in bronchial cells obtained from smoking individuals.

The binding efficiency of polyclonal and monoclonal antibodies to DNA modified with benzo[a]pyrene diol epoxide is dependent on the level of modification. Implications for quantitation of benzo[a]pyrene-DNA adducts in vivo.

A number of polyclonal antibodies specific for DNA modified with (+/-)trans-7,8-dihydroxy-anti-9,10-epoxy-7,8,9,10-tetrahydrobenzo[a]pyre ne (BPDE) were obtained from the sera of New Zealand white rabbits immunized with BPDE-DNA, complexed with methylated bovine serum albumin (mBSA). Monoclonal antibodies were developed by fusion of mouse myeloma cells with spleen cells isolated from BALB/c mice immunized with the same complex of BPDE-DNA and mBSA. These antibodies have been characterized for specificity in a highly sensitive, enzyme-linked immunosorbent assay (ELISA). All antibodies showed a very high affinity for single-stranded BPDE-DNA, but had lower affinity towards native BPDE-DNA. The affinity for the free mononucleoside BPDE-dG was at least 100-fold lower than that for BPDE-DNA, and no affinity was detected for BP tetrols or DNA modified with N-acetoxy-N-acetyl-2-aminofluorene. A high cross reactivity was observed with DNA modified with (+/-)-trans-1,2-dihydroxy-anti-3,4-epoxy-1,2,3,4-tetrahydrochrysene++ +. Using five different antibodies, monoclonal or polyclonal, we observed that the antibody affinity for BPDE-DNA was dependent on the level of modification; in the competitive ELISA as little as 4 fmol BPDE-DNA (50 pmol/micrograms) was sufficient for 50% inhibition with our best antisera, but 17 fmol of the adduct was required when [3H]BPDE-DNA of low modification (1-10 fmol/micrograms) was used as inhibitor. When samples of [3H]BP-DNA isolated from the livers of mice, treated i.p. with different doses of [3H]BP were examined by competitive ELISA and calibrated with [3H]BPDE-DNA of low modification (1-10 fmol/micrograms), binding values calculated from the immunoassay were in good agreement with those obtained from radioactivity measurements. In contrast, when this DNA was quantitated in competitive ELISA using highly modified BPDE-DNA as standards, values by ELISA were 20-40% of those obtained by radioactivity. These results indicate that the use of serially diluted BPDE-DNA of high modification as standard competitor in the ELISA will lead to erroneous results in the measurement of adducts in DNAs modified to a low extent (biological samples). The property of antisera specific for BP-DNA, recognizing highly modified DNA more efficiently than DNA modified to a low extent, may be common to all antisera elicited against highly modified DNA immunogens. Therefore we conclude that antibody affinity must be tested also with DNA samples of low modification, obtained either in vitro or in vivo.