Photochemistry of the furan-side 8-methoxypsoralen-thymidine monoadduct inside the DNA helix. Conversion to diadduct and to pyrone-side monoadduct

We have studied the photochemical reactions of 8-methoxypsoralen (8-MOP) with calf thymus DNA. Analysis of the photoproducts formed was carried out by enzymatic digestion of the 8-MOP-modified DNA, followed by HPLC separation of photoadducts by high-pressure liquid chromatography (HPLC). The 4',5' (furan-side) monoadduct of 8-MOP bound to thymidine is converted to cross-linked thymidine-8-MOP-thymidine diadduct by 341.5 nm light with a quantum yield of 0.028 +/- 0.004. This is 4 times greater than the quantum yield for initial adduct formation (0.0065 +/- 0.0004). When low levels of 8-MOP are covalently bound to DNA by using 397.9 nm light, less than 10% of the adducts formed are diadducts yet nearly 70% are in 5'-TpA cross-linkable sites. The furan-side monoadducts in these sites can subsequently be converted to diadduct or to a lesser extent 3,4 (pyrone-side) monoadduct.     

Detection of Adriamycin-DNA adducts by accelerator mass spectrometry at clinically relevant Adriamycin concentrations

Limited sensitivity of existing assays has prevented investigation of whether Adriamycin-DNA adducts are involved in the anti-tumour potential of Adriamycin. Previous detection has achieved a sensitivity of a few Adriamycin-DNA adducts/10(4) bp DNA, but has required the use of supra-clinical drug concentrations. This work sought to measure Adriamycin-DNA adducts at sub-micromolar doses using accelerator mass spectrometry (AMS), a technique with origins in geochemistry for radiocarbon dating. We have used conditions previously validated (by less sensitive decay counting) to extract [(14)C]Adriamycin-DNA adducts from cells and adapted the methodology to AMS detection. Here we show the first direct evidence of Adriamycin-DNA adducts at clinically-relevant Adriamycin concentrations. [(14)C]Adriamycin treatment (25 nM) resulted in 4.4 +/- 1.0 adducts/10(7) bp ( approximately 1300 adducts/cell) in MCF-7 breast cancer cells, representing the best sensitivity and precision reported to date for the covalent binding of Adriamycin to DNA. The exceedingly sensitive nature of AMS has enabled over three orders of magnitude increased sensitivity of Adriamycin-DNA adduct detection and revealed adduct formation within an hour of drug treatment. This method has been shown to be highly reproducible for the measurement of Adriamycin-DNA adducts in tumour cells in culture and can now be applied to the detection of these adducts in human tissues.     

Real-time fluorescence-based detection of furanocoumarin photoadducts of DNA

Real-time fluorescence detection systems were adapted to identify DNA adducts formed by photogenotoxic phytochemicals. Two assays were developed: the first was based on quantitative polymerase chain reaction (qPCR) while the second used thermal denaturation and renaturation (D-R). Both assays employed yeast DNA, the fluorescent dye SYBR Green and a real-time PCR thermocycler. The furanocoumarins 8-methoxypsoralen (8-MOP), 5-methoxypsoralen (5-MOP), psoralen, angelicin and imperatorin, and the furanochrome khellin, were tested for adduct forming ability with up to 2 h of UVA light exposure (lambda = 320-400 nm). The known bifunctional compounds, 8-MOP, 5-MOP and psoralen, were inferred to form biadducts here based on both D-R and qPCR assays, as expected from previous research. The known monofunctional compound angelicin was used as a negative control and did not form biadducts based on either assay. Two compounds of unknown functional specificity, imperatorin and khellin, were determined to be positive and negative for biadduct activity, respectively. Detection of biadducts with 8-MOP, 5-MOP, psoralen and imperatorin, but not angelicin or khellin, was further verified by temperature gradient gel electrophoresis. The fluorescence methods improve and expand upon existing assays to monitor DNA adducts.     

Detection of Adriamycin–DNA adducts by accelerator mass spectrometry at clinically relevant Adriamycin concentrations

Limited sensitivity of existing assays has prevented investigation of whether Adriamycin–DNA adducts are involved in the anti-tumour potential of Adriamycin. Previous detection has achieved a sensitivity of a few Adriamycin–DNA adducts/10⁴ bp DNA, but has required the use of supra-clinical drug concentrations. This work sought to measure Adriamycin–DNA adducts at sub-micromolar doses using accelerator mass spectrometry (AMS), a technique with origins in geochemistry for radiocarbon dating. We have used conditions previously validated (by less sensitive decay counting) to extract [¹⁴C]Adriamycin–DNA adducts from cells and adapted the methodology to AMS detection. Here we show the first direct evidence of Adriamycin–DNA adducts at clinically-relevant Adriamycin concentrations. [¹⁴C]Adriamycin treatment (25 nM) resulted in 4.4 ± 1.0 adducts/10⁷ bp (~1300 adducts/cell) in MCF-7 breast cancer cells, representing the best sensitivity and precision reported to date for the covalent binding of Adriamycin to DNA. The exceedingly sensitive nature of AMS has enabled over three orders of magnitude increased sensitivity of Adriamycin–DNA adduct detection and revealed adduct formation within an hour of drug treatment. This method has been shown to be highly reproducible for the measurement of Adriamycin–DNA adducts in tumour cells in culture and can now be applied to the detection of these adducts in human tissues.     

Repair of 8-methoxypsoralen monoadducts in mouse lymphoma cells

Studies of the repair of DNA lesions at biologically important doses is extremely difficult for most mutagens. With 8-methoxypsoralen (8-MOP) plus longwave ultraviolet light (UVA) as the lesion-inducing agent, however, it is easy to manipulate the relative frequency of different DNA adducts by means of a special experimental protocol (the tap-and-test protocol) and this can be used to measure repair of DNA adducts. Three classes of photoadducts are produced by 8-MOP plus UVA treatment: 3,4-cyclobutane monoadducts, 4',5'-cyclobutane monoadducts, and 8-MOP-DNA interstrand crosslinks. A monoadduct is formed when a photoactivated 8-MOP molecule reacts with a pyrimidine base. An 8-MOP-DNA interstrand crosslink is formed when an existing monoadduct is photoactivated to react with another pyrimidine base on the opposite DNA strand. Thus monoadducts are formed by absorption of one photon of light and crosslinks by absorption of two. In the tap-and-test experiments, cells were exposed to UVA in the presence of 8-MOP and then re-exposed to UVA in the absence of free 8-MOP so that only crosslinks can be produced by the second UVA treatment. By means of this technique we have previously shown that DNA crosslinks are much more effective than monoadducts at producing chromosomal damage (sister-chromatid exchanges and micronuclei) but not mutations (Liu-Lee et al., 1984). If L5178Y mouse lymphoma cells were able to remove monoadducts, incubation prior to the second UVA treatment should lead to decreases in the effect of re-irradiation, because fewer monoadducts would be available for crosslink formation. In this way, we have found that psoralen monoadducts are repaired in these cells and that about 70% of those capable of crosslink formation are removed or otherwise made unavailable for crosslink formation in 6 h.     

Monoclonal antibodies to DNA modified by 8-methoxypsoralen and ultraviolet A light.

A panel of monoclonal antibodies have been developed which specifically recognize DNA modified by 8-methoxypsoralen (8-MOP) and ultraviolet A light (320-400 nm) (UVA). These antibodies have been characterized as to sensitivity and specificity by an enzyme linked immunosorbent assay (ELISA). In a competitive ELISA with the most sensitive antibody, 50% inhibition of antibody binding occurred at 17 fmole 8-MOP-DNA photo adducts. One adduct per 10(7) bases could be reliably detected. There was also some antibody cross-reactivity with DNAs modified by 4' aminomethyl-4, 5, 8-trimethylpsoralen and 4', 5-dimethylangelicin as well as DNA isolated from cells treated with 8-MOP and UVA. The primary specificity of one of the antibodies was shown to be the 4', 5' thymine monoadduct by competitive inhibition studies using HPLC fractions of an enzymatic digest of 8-MOP poly(dA-dT) . poly(dA-dT). These antibodies should allow the quantitation of adduct levels in various in vitro systems as well as humans exposed clinically to 8-MOP and UVA.     

Method for estimation of benzo[a]pyrene DNA adducts.

Polycyclic aromatic hydrocarbons, e.g., benzo[a]pyrene (B(a)P) are known carcinogens/mutagens. These compounds may be metabolized by the P450 mixed function monooxygenase to more nucleophilic compounds which may form adducts to the cellular macromolecules, e.g., DNA, RNA, and proteins. We have used synchronous fluorescence scanning for the assay of DNA adduct formation. In our earlier work with in vitro exposed human lymphocytes we estimated the adduct formation (femtomoles B(a)P per microgram DNA) to be higher than that estimated by other workers. We suggested that this difference may be related to the DNA isolation method used. In order to elucidate these differences we compared DNA adduct formation in human lymphocytes where DNA was isolated by the two different methods, i.e., using phenol extraction or the Gene Clean method. The data demonstrate that the phenol extraction procedure gives a yield of adducts per microgram DNA lower than that obtained by the Gene Clean method. The principle of the Gene Clean method for DNA isolation is protein denaturation by means of NaI followed by catching of DNA by absorption on silica particles. In contrast, the phenol extraction method is based upon phenol-mediated denaturation of proteins in the cell lysate leaving the hydrophilic nucleotides in the aqueous phase. However, during adduct formation more lipophilic adducts derived from DNA may redistribute between the aqueous phase and the phenol phase. In support of this theory we found higher adduct concentration per microgram DNA by the Gene Clean method 40 to 60 times than that found by the phenol method.(ABSTRACT TRUNCATED AT 250 WORDS)     

Effects of 8-methoxypsoralen and ultraviolet light A on EGF receptor (HER-1) expression

Activation of psoralens by ultraviolet light irradiation at 308-400 nm (UVA) is used in the photochemical treatment of psoriasis. While the major effect of this activation is the formation of DNA adducts, it was recently demonstrated that psoralens can also bind to specific saturable high affinity cellular receptors, and that this is associated with inhibition of epidermal growth factor (EGF)-receptor binding. In view of these findings, we have examined whether 8-methoxy-psoralen (8-MOP) itself, or in combination with UVA, influences expression of the human EGF-receptor gene ("HER-1") in a human keratinocyte cell line. We have found that 8 MOP alone, and to a lesser extent UVA, induce a striking increase in cellular levels of HER-1 RNA. The combination of 8-MOP with UVA produces less induction of HER-1 RNA than that obtained with 8-MOP alone. We suggest, therefore, that this effect of 8-MOP is not due to DNA damage, but may reflect a separate effect of this compound on receptor-mediated signal transduction.     

W-reactivation and W-mutagenesis in lambda and T7 bacteriophages: a comparative study of the action of ultraviolet radiation (254 nm) and of the photosensitizing agents, 8-methoxypsoralen and angelicin

Monoadducts and interstrand cross-links are formed in DNA after psoralen plus light treatment of bacteriophage lambda . Survival and clear plaque mutation frequency of lambda after photosensitization with 8-methoxypsoralen (8-MOP) are increased when the wild type host is slightly UV-irradiated (W-reactivation and W-mutagenesis). The recA13, lexA1 and uvrA6 mutations block W-reactivation and W-mutagenesis of lambda treated with 8-MOP plus light. Using the technique of "repeated irradiation" we showed that the mutagenic effect of 8-MOP plus light treatment on phage is due mainly to formation of cross-links in DNA. The mutagenic activity of monoadducts had been studied by using angular furocoumarin, angelicin which forms mainly monoadducts in DNA. Upon W-mutagenesis of phage lambda treated with angelicin plus light a high mutagenic effect is observed. The results indicate that the mutagenic activity of monoadducts is 15-20 fold slower as compared to that of cross-links. W-reactivation and W-mutagenesis of UV-irradiated (254 nm) bacteriophage lambda are also observed after 8-MOP plus light treatment of Escherichia coli uvrA and wild type hosts. It is possible that the difference in mutagenic activity of psoralen adducts could depend on the repair mechanism of adducts: cross-links repair in bacterial and lambda DNA is controlled by lexA gene (error-prone SOS-repair mechanism), while monoadducts can be efficiently repaired by error-free excision and recombination.     

DNA photobinding of 7-methylpyrido[3,4-c]psoralen and 8-methoxypsoralen. Effects on macromolecular synthesis, repair and survival in cultured human cells

The photobinding to DNA of tritiated 7-methylpyrido[3,4-c]psoralen (MPP), a recently synthesized monofunctional compound of therapeutical interest, and of 8-methoxypsoralen (8-MOP) was determined in cultured normal human fibroblasts. Employing compounds at 10(-6) M, MPP photobinds approximately 11 times more efficiently than 8-MOP: one molecule is fixed respectively per 7.5 X 10(4) or 8.1 X 10(5) base pairs/kJ . m-2 of 365-nm radiation (UVA). Removal of bound material from DNA is slow and limited in 48-72 h of post-treatment incubation to 30-40% of initial adducts formed by MPP and to 50-60% of those of 8-MOP. For equivalent photobinding MPP and 8-MOP induce similar inhibitions of DNA synthesis. However, the recovery of DNA synthesis during post-treatment incubation is lower after photoaddition of MPP than after that of 8-MOP. MPP also exerts a much higher lethal effect than 8-MOP: one lethal hit corresponds to about 4400 and to 19,900 adducts per cell respectively. Alkaline elution experiments confirmed the monofunctional nature of MPP and indicated that in MPP-damaged cells DNA breaks accumulate with time of post-treatment incubation. In contrast, after photoaddition of 3-carbethoxypsoralen (3-CPs), another monofunctional furocoumarin, or irradiation with 254-nm UV, DNA breaks are induced only transiently. In 8-MOP-treated cells, DNA cross-links appear to be partially repaired. In conclusion, MPP monoadducts turn out to constitute more cytotoxic lesions than 8-MOP mono- and bi-adducts.     

32P-postlabelling detection of aromatic DNA adducts in peripheral blood lymphocytes from aluminium production plant workers

Aluminium production plant workers are exposed to a great number of airborne polycyclic aromatic hydrocarbons and epidemiological studies suggest that these workers are at increased risk of lung and bladder cancer. Blood samples from 46 workers at 2 primary aluminium plants and from 29 occupationally unexposed control individuals were analysed. DNA was isolated from the peripheral blood lymphocytes and aromatic DNA adducts were detected by 32P-postlabelling assay using the nuclease P1 digestion procedure for the enrichment of the adducts. The total levels of DNA adducts of exposed individuals varied from the detection limit of about 0.5 adducts/10(8) nucleotides up to 7.1 adducts/10(8) nucleotides and control adduct levels were up to 2.42 adducts/10(8) nucleotides. There was no significant difference between the mean adduct levels of the control group and of the individuals of one plant. However, the mean DNA adduct level obtained from workers of the second plant was significantly higher than that of the controls (p less than 0.001) and of the first plant (p less than 0.01), respectively. This difference can be attributed to differences in the design of technology and different levels of exposure at the 2 plants. The results of this study encourage further investigations of the use of peripheral white blood cells as marker cells and of 32P-postlabelling analysis for monitoring occupational exposure to mixtures of environmental carcinogenic pollutants.     

Preferential repair of N-acetoxy-acetylaminofluorene lesions in the nuclease-hypersensitive region of simian virus 40

We exposed simian virus 40-infected CV-1 monkey cells to the carcinogen N-acetoxy-acetylaminofluorene and monitored the removal of lesions from cellular DNA and from various regions of viral DNA. Exposure to 5.5 microM 3H-labeled carcinogen produced 20-80 adducts per 10(6) bases in cellular DNA in different experiments. The initial adduct concentration in viral DNA was always approximately half that in cellular DNA. At various times after treatment, cellular and viral DNA, and restriction fragments of viral DNA, were purified and examined for adduct density. Independent of the initial adduct concentration three rates of repair were observed. Cellular DNA was repaired at the lowest rate. Viral DNA was repaired about 50% more rapidly than was cellular DNA isolated from the same carcinogen-treated monolayers. Within the viral DNA a 366-base pair region containing the major nuclease-hypersensitive site was repaired at twice the rate of the rest of the viral genome. This region contains regulatory sequences that govern the initiation of DNA replication and viral gene expression. As reported previously this region was initially modified 1.71 +/- 0.20-fold higher than expected from its guanine content. Selective repair diminished the extent of hypermodification of this region by 6.0 +/- 2.1% per hour, partly compensating for the higher initial level of adducts.     

Modification of DNA by mitomycin C in cancer patients detected by 32P-postlabeling analysis

DNA adducts of mitomycin C (MMC) were detected by 32P-postlabeling analysis in both surgical specimens and an autopsy sample of the liver of patients with hepatocellular carcinoma who had received chemotherapy with MMC. Four kinds of adducts were detected in all 6 patients treated with MMC. These adducts had identical chromatographic mobilities to those of adducts in the liver of rats treated with MMC, but 1 additional adduct was detected in rat liver. In patients treated with MMC, about 3 adducts/10(8) nucleotides were found 4 days after MMC treatment, and 1 adduct/10(8) nucleotides 14 days after treatment and the latter level was maintained for up to 56 days. MMC-DNA adducts were also detected in peripheral blood leukocytes from a patient 1 and 7 days after MMC treatment, at levels of 1 and 0.6 adduct/10(8) nucleotides, respectively. These results suggest the tumor-initiating activity of MMC in humans.     

The use of Taq DNA polymerase to determine the sequence specificity of DNA damage caused by cis-diamminedichloroplatinum(II), acridine-tethered platinum(II) diammine complexes or two analogues.

cis-Diamminedichloroplatinum(II) (cisplatin) forms adducts with DNA. The sequence specificity of formation of cisplatin adducts with plasmid DNA was investigated using Taq DNA polymerase. This procedure involved the extension of an oligonucleotide primer by Taq DNA polymerase up to the cisplatin adduct. Using thermal cycling, this process is repeated many times in order to amplify the signal. The products of this linear amplification can then be examined on DNA sequencing gels, and the sequence specificity of cisplatin adduct formation can be determined to the exact base pair. In the pUC8 plasmid, the sequences that produced the most intense damage sites (as determined by densitometry) were runs of two or more Gs. Adducts could also be detected at GA, AG, and GC dinucleotides. Four other cisplatin analogues were also tested in the system. Two of these analogues contained an attached intercalating chromophore, and the strong damage with these compounds was similar to that found for cisplatin, but the medium and weak damage tended to be different. Weak damage was also detected with trans-diamminedichloroplatinum(II). With this compound, a large number of the damage sites were at the CG dinucleotide. This technique represents a simple, accurate, and quick method for determining the sequence specificity of damage for a cisplatin analogue in any DNA sequence.     

Comparative study of the lethal effects of near-UV light (360 nm) and 8-methoxypsoralen plus near-UV on plasmid DNA

We have studied the lethality produced on pBR322 by near-UV radiation and by 8-Methoxypsoralen plus near-UV (PUV treatment). Samples of pBR322 DNA were irradiated with increasing fluences of 360 nm-light either in the absence or presence of 400 molecules of 8-Methoxypsoralen (8-MOP) per plasmid molecule. We have estimated to what extent the global lethality of PUVA treatment is due to the presence of psoralen adducts in DNA or to radiation itself. In order to analyse the involvement of DNA repair mechanisms in the removal of plasmid lesions, several strains of E. coli (differing in their repair capacities) were used as recipients of the treated plasmids. Results showed that excision and recombination participate in the repair of near-UV-induced plasmid lesions. Repair of PUV-induced lesions showed an even greater requirement of the excision pathway. Besides, a slight increase on plasmid mutation frequencies was observed after near-UV or PUV treatment in wild type and uvrA cells. Estimation of the contribution of 8-MOP to the global lethality of PUV treatment showed that only the excision pathway was involved in removing psoralen adducts from plasmid DNA, suggesting the involvement of the recombinational pathway in the repair of near-UV-derived lesions.     

PUVA (8-methoxy-psoralen plus ultraviolet A) induces the formation of 8-hydroxy-2'-deoxyguanosine and DNA fragmentation in calf thymus DNA and human epidermoid carcinoma cells.

The objective of this study is to investigate if 8-methoxy-psoralen (8-MOP) plus ultraviolet A (UVA) radiation (PUVA) induces oxidative DNA damage. When calf thymus DNA was incubated with 8-MOP and irradiated with UVA (335-400 nm), the level of 8-hydroxy-2'-deoxyguanosine (8-OHdG) was substantially increased by approximately 6-fold. Formation of 8-OHdG proportionally correlated with both UVA fluence and 8-MOP concentrations. Human epidermoid carcinoma cells were incubated with 10 microg 8-MOP per milliliter, followed by irradiation of 25 kJ/m2 UVA. The level of 8-OHdG increased by nearly 3-fold in PUVA-treated cells compared to 8-MOP and UVA controls. The formation of 8-OHdG correlated with DNA fragmentation as determined by spectrofluorometry. To investigate the reactive oxygen species (ROS) involved in PUVA-induced oxidative DNA damage, less or more specific ROS quenchers were added to DNA solution prior to PUVA treatment. The results showed that only sodium azide and genistein significantly quenched PUVA-induced 8-OHdG, whereas catalase, superoxide dismutase, and mannitol exhibited no effect. The quencher study with cultured cells indicated that N-acetyl-cysteine and genistein protected oxidative DNA damage as well as DNA fragmentation by PUVA treatment. Our studies show that PUVA treatment is able to induce the formation of 8-OHdG in purified DNA and cultured cells and suggest that singlet oxygen is the principle reactive oxygen species involved in oxidative DNA damage by PUVA treatment.     

Excision of benzo[a]pyrene diol epoxide I adducts from nucleosomal DNA of confluent normal human fibroblasts

The formation and removal of covalent adducts of racemic 7 beta, 8 alpha-dihydroxy-9 alpha,10 alpha-epoxy-7,8,9,10-tetrahydrobenzo[a]pyrene (BPDE I) was studied in nucleosomal DNA of confluent cultures of normal human fibroblasts (NF). For this purpose NF were prelabeled in their DNA with [14C]-thymidine and treated with [3H]BPDE I. The adducts were composed of 77% (7R)-N2-(7 beta, 8 alpha, 9 alpha-trihydroxy-7,8,9,10-tetrahydrobenzo[a]pyrene-10-yl)deoxyguanosine, 12% of the corresponding 7S-enantiomer and of minor amounts of adducts to cytosine and adenine. The adduct composition did not change significantly in 24-h post treatment incubation. Bulk mononucleosomes were prepared from micrococcal nuclease digested nuclei and their DNA analyzed by gel electrophoresis. The adduct concentrations were determined in 145 base pair (b.p.) nucleosomal core-DNA, 165 b.p. chromatosomal DNA and in total nuclear DNA. From these data the concentration in nucleosomal linker-DNA was calculated. The initial adduct distribution was non-random and 6.3 times higher in 47 b.p. linker-DNA relative to 145 b.p. core-DNA and 9.2 times higher in 27 b.p. linker-DNA relative to 165 b.p. chromatosomal DNA. Adduct removal was very rapid during the first 8 h and more efficient from linker-DNA than from core-DNA. After this early phase the adducts located in 145 b.p. core-DNA became refractory to further excision and represent a major fraction of the adducts persisting in DNA of NF over a prolonged period. In contrast, further adduct removal was observed from nucleosomal linker-DNA.     

Polycyclic aromatic hydrocarbon-DNA adducts in cervix of women infected with carcinogenic human papillomavirus types: an immunohistochemistry study

Among women infected with carcinogenic human papillomavirus (HPV), there is a two- to five-fold increased risk of cervical precancer and cancer in women who smoke compared to those who do not smoke. Because tobacco smoke contains carcinogenic polycyclic aromatic hydrocarbons (PAHs), it was of interest to examine human cervical tissue for PAH-DNA adduct formation. Here, we measured PAH-DNA adduct formation in cervical biopsies collected in follow-up among women who tested positive for carcinogenic HPV at baseline. A semi-quantitative immunohistochemistry (IHC) method using antiserum elicited against DNA modified with r7,t8-dihydroxy-t-9,10-oxy-7,8,9,10-tetrahydrobenzo[a]pyrene (BPDE) was used to measure nuclear PAH-DNA adduct formation. Cultured human cervical keratinocytes exposed to 0, 0.153, or 0.331microM BPDE showed dose-dependent increases in r7,t8,t9-trihydroxy-c-10-(N(2)deoxyguanosyl)-7,8,9,10-tetrahydro-benzo[a]pyrene (BPdG) adducts. For BPdG adduct analysis, paraffin-embedded keratinocytes were stained by IHC with analysis of nuclear color intensity by Automated Cellular Imaging System (ACIS) and, in parallel cultures, extracted DNA was assayed by quantitative BPDE-DNA chemiluminescence immunoassay (CIA). For paraffin-embedded samples from carcinogenic HPV-infected women, normal-appearing cervical squamous epithelium suitable for scoring was found in samples from 75 of the 114 individuals, including 29 cases of cervical precancer or cancer and 46 controls. With a lower limit of detection of 20 adducts/10(8) nucleotides, detectable PAH-DNA adduct values ranged from 25 to 191/10(8) nucleotides, with a median of 75/10(8) nucleotides. PAH-DNA adduct values above 150/10(8) nucleotides were found in eight samples, and in three samples adducts were non-detectable. There was no correlation between PAH-DNA adduct formation and either smoking or case status. Therefore, PAH-DNA adduct formation as measured by this methodology did not appear related to the increased risk of cervical precancer and cancer among carcinogenic HPV-infected smokers.     

Dose responses for DNA adduct formation in tissues of rats and mice exposed by inhalation to low concentrations of 1,3-[2,3-[(14)C]-butadiene

Male Sprague-Dawley rats and B6C3F1 mice were exposed to either a single 6h or a multiple (5) daily (6h) nose-only dose of 1,3-[2,3-(14)C]-butadiene at exposure concentrations of nominally 1, 5 or 20 ppm. The aim was to compare the results with those from a similar previous study at 200 ppm. DNA isolated from liver, lung and testis of exposed rats and mice was analysed for the presence of butadiene related adducts, especially the N7-guanine adducts. Total radioactivity present in the DNA from liver, lung and testis was quantified and indicated more covalent binding of radioactivity for mouse tissue DNA than rat tissue DNA. Following release of the depurinating DNA adducts by neutral thermal hydrolysis, the liberated depurinated DNA adducts were measured by reverse phase HPLC coupled with liquid scintillation counting. The guanine adduct G4, assigned as N7-(2,3,4-trihydroxybutyl)- guanine, was the major adduct measured in liver, lung and testis DNA in both rats and mice. Higher levels of G4 were detected in all mouse tissues compared with rat tissue. The dose-response relationship for the formation of adduct G4 was approximately linear for all tissues studied for both rats and mice exposed in the 1-20 ppm range. The formation of G4 in liver tissue was about three times more effective for mouse than rat in this exposure range. Average levels of adduct G4 measured in liver DNA of rats and mice exposed to 5 x 6 h 1, 5 and 20 ppm 1,3-[2,3-(14)C]-butadiene were, respectively, for rats: 0.79 +/- 0.30, 2.90 +/- 1.19, 16.35 +/- 4.8 adducts/10(8) nucleotides and for mice: 2.23 +/- 0.71, 12.24 +/- 2.15, 48.63 +/- 12.61 adducts/10(8) nucleotides. For lung DNA the corresponding values were for rats: 1.02 +/- 0.44, 3.12 +/- 1.06, 17.02 +/- 4.07 adducts/10(8) nucleotides, and for mice: 3.28 +/- 0.32, 14.04 +/- 1.55, 42.47 +/- 13.12 adducts/10(8) nucleotides. Limited comparative data showed that the levels of adduct G4 formed in liver and lung DNA of mice exposed to a single exposure to butadiene in the present 20 ppm study and earlier 200 ppm study were approximately directly proportional across dose, but this was not observed in the case of rats. From the available evidence it is most likely that adduct G4 was formed from a specific isomer of the diol-epoxide metabolite, 3,4-epoxy-1,2-butanediol rather than the diepoxide, 1,2,3,4-diepoxybutane. Another adduct G3, possibly a diastereomer of N7-(2,3,4-trihydroxybutyl)-guanine or most likely the regioisomer N7-(1-hydroxymethyl-2,3-dihydroxypropyl)-guanine, was also detected in DNA of mouse tissues but was essentially absent in DNA from rat tissue. Qualitatively similar profiles of adducts were observed following exposures to butadiene in the present 20 ppm study and the previous 200 ppm study. Overall the DNA adduct levels measured in tissues of both rats and mice were very low. The differences in the profiles and quantity of adducts seen between mice and rats were considered insufficient to explain the large difference in carcinogenic potency of butadiene to mice compared with rats.     

Monitoring human exposure to carcinogens by DNA adduct measurement

Sensitive immunologic techniques are now available for the detection and quantitation of carcinogen-DNA adducts. We have developed a number of specific monoclonal antibodies which recognize DNA modified by particular carcinogens, including benzo[a]-py-rene, aminopyrene, 8-methoxypsoralen plus UVA light and vinyl chloride. These antibodies can be used in competitive enzyme-linked immunosorbent assays to quantitate adducts in DNA isolated from biological samples. Samples from treated animals as well as from humans with occupational or environmental exposure to carcinogens have been studied. In addition, antibodies can be used in indirect immunohistochemical studies to localize adduct formation in various tissues or cell types.Abbreviations BPDE-1 7,8-dihydroxy-9,10-epoxy-7,8,9,10-tetrahydrobenzo[a]pyrene - CTCL Cutaneous T cell lymphoma - ELISA enzyme linked immunosorbent assay - 8-MOP 8-methoxypsoralen - PUVA 8-MOP plus UVA irradiation