Ultrasensitive enzymatic radioimmunoassay (USERIA) detects femtomoles of acetylaminofluorene-DNA adducts

USERIA is a modification of radioimmunoassay and enzyme-linkedimmunosorbent assay. When compared to these assays, USERIA is60- and 10-fold, respectively, more sensitive for the detectionof ad-ducts in DNA modified by the chemical carcinogen, 2-acetylaminofluorene(AAF). The specific antigen-antibody reaction is enzymaticallyamplified by an anti-IgG alkaline phosphatase conjugate whichconverts the substrate, [³H]adenosine-5'-mono-phosphate, to[³H]adenosine. As few as 2 fmol of AAF-DNA adducts can be detectedby a competitive USERIA assay and less than 3 fmol of the specificadduct, N-(deoxyguanosine-8-yl)-AAF, can be detected using anon-competitive USERIA approach. The sensitivity of USERIA shouldaid efforts to measure carcinogen-DNA adducts in biologicalspecimen samples from humans and experimental animals.     

Induction and removal of cisplatin-DNA adducts in human cells in vivo and in vitro as measured by immunochemical techniques

The same spectrum of cisplatin adducts was detected in DNA isolated from white blood cells of a cisplatin-treated cancer patient as had been found in cisplatin-treated DNA in vitro. The adducts were quantified in femtomole amounts by competitive enzyme-linked immunosorbent assay (ELISA) with three antisera raised against synthetic cisplatin-containing (oligo)nucleotides. For this assay, DNA samples digested with nucleases were fractionated by ion-exchange chromatography; the fractions were used as inhibitors of antibody binding. Determinations of the main adduct formed, cis-Pt(NH3)2d(pGpG), in patients immediately after a first treatment with equal doses of cisplatin showed interindividual differences in the platination levels of the white blood cells. These differences were found to correlate with those found after in-vitro exposure to cisplatin of blood samples taken from patients before treatment. In vivo, about 75% of the adducts formed after the first treatment were removed within 24 h. During a five-day course, the amounts of the main adduct increased after the first three administrations; no increase was seen on day 4 or 5. By day 6, considerable removal of adducts had occurred. Analysis of the formation and repair of the cis-Pt(NH3)2d(pGpG) adducts in cultured cells, i.e., human fibroblasts with different DNA repair capacities and one bladder and two testicular human cancer cell lines, indicated that both the amounts of adducts formed and the ability of the cells to repair the adducts can differ. These differences appear to determine the susceptibility of the cells for the cytotoxic action of cisplatin.     

Detection of N-hydroxy-2-acetylaminofluorene--DNA adducts in rat liver measured by radioimmunoassay

Antibodies to imidazole ring opened derivatives of alkali treatedN-(guanosin-8-yl)-2-aminofluorene (1-[6-(2, 5-diamino-4-oxopyrimidinyl-N⁶-riboside)]-3-(2-fluorenyl)ureawere elicited in rabbits by immunization with a conjugate betweenthe ring-opened derivative and bovine serum albumin. The specificityof the antibodies was studied by radio-immunoassay. These antibodiesand antibodies to N-(guanosin-8-yl)-N-acetyl-2-aminofluorenewere used to titrate the adducts formed in liver DNA of ratstreated with N-hydroxy-2-acetylaminofluorene. The ring-openedderivatives represent 10% of the dGuo-C8-adducts.     

Formation and removal of dibenzo[a,e]fluoranthene-DNA adducts in mouse embryo fibroblasts

In vivo binding of dibenzo[a, e]fluoranthene (DBF) to mouseembryo fibroblast DNA was compared with that observed previouslyin vitro on calf thymus DNA incubated with mouse liver microsomes.The h.p.l.c. elution patterns of the adducts formed by DBF metaboliteswith DNA and obtained in vivo at the optimal exposure time of42–48 h were qualitatively very similar to the patternsobtained in vitro, but their amplitude was quantitatively reduced.There are two striking differences between the in vivo and invitro results. Firstly, the most polar peak A, very abundantin vitro, was absent in vivo. Secondly, the reactivity of thetwo major proximate metabolites of DBF, the bay and pseudo-bayregion dihydrodiols, was very different in intact cells comparedwith the results in vitro. When incubated in vitro, pseudo-bayregion dihydrodiol DBF was twice as reactive as bay region dihydrodiolDBF. The opposite reactivities were observed in vivo. The majorDBF-DNA adducts formed in vivo were collected in the peaks E,B and C. The predominant peak E contained DNA adducts of bothbay and pseudo-bay region dihydrodiolepoxides which are themajor ultimate metabolites of DBF in vivo and in vitro. Theother two prominent peaks B and C contained DNA adducts of 3-hydroxyDBF pseudo-bay region dihydrodiolepoxide and the 7-hydroxy DBFbay region dihydrodiolepoxide, respectively. After adduct formation,post incubation of fibroblasts for a further 48 h, in the absenceof DBF, eliminated half the amount of adducts present. PeakB adducts were repaired more efficiently than those of peaksE, C D and F. The carcinogenic initiating activity of DBF appearsto be a complex process in which several DNA adducts play arole.     

Lack of mutagenicity of acrolein-induced DNA adducts in mouse and human cells

Acrolein is an endogenous metabolite and a ubiquitous environmental pollutant. Recently, it has been suggested that acrolein is a major etiologic agent for tobacco smoking-related lung cancer. Despite the known DNA-damaging effects of acrolein, its mutagenicity to mammalian cells remains uncertain. We have investigated acrolein-induced DNA damage in relation to mutagenesis, with special focus on DNA repair, in mouse and human cells. We mapped the formation of acrolein-induced DNA adducts and the kinetics of repair of the induced lesions in the cII transgene, the mutational target, in acrolein-treated transgenic mouse fibroblasts. Acrolein-DNA adducts were formed preferentially at specific nucleotide positions, mainly at G:C base pairs, along the cII transgene. The induced acrolein-DNA adducts were moderately resistant to DNA repair. Quantification of cII mutant frequency in acrolein-treated cells, however, revealed that acrolein was not mutagenic to these cells at doses sufficient to produce DNA adducts. Determination of supF mutant frequency in DNA repair-proficient and DNA repair-deficient human fibroblasts transfected with acrolein-treated plasmids confirmed a lack of acrolein mutagenicity. Because CpG methylation may intensify acrolein-DNA adduction, we examined whether the extent of CpG methylation in the supF gene can determine acrolein-induced mutagenesis in human cells. Enhancement of acrolein-DNA adduction by methylating CpGs in the supF sequence did not elicit a mutagenic response in human fibroblasts, however. We conclude that acrolein is not mutagenic to mouse and human fibroblasts, regardless of DNA repair capacity or methylation status of CpGs, possibly because of a highly accurate replication bypass of the induced lesions.     

Formation of mitoxantrone adducts in human tumor cells: potentiation by AN-9 and DNA methylation

The ability of mitoxantrone to form DNA adducts was investigated in a series of human tumor cell lines consisting of human cervical cancer (HeLa), human breast cancer (MCF-7), and human neuroblastoma (IMR-32) cells. The mitoxantrone-resistant human promyelocytic leukemia cell line HL60/MX2 was also compared to the parental cell line HL60 in terms of adduct formation in cellular DNA, RNA, and protein. DNA adduct formation detected using [14C]mitoxantrone as a single agent occurred at very low levels but addition of the formaldehyde-releasing prodrug AN-9 (pivaloyloxymethyl butyrate) increased adduct formation considerably in all cell lines tested. Adduct formation increased when increasing ratios of AN-9 were used, and were observed at maximal levels when AN-9 addition was 4 h after the addition of mitoxantrone. However, low levels of adducts were observed when AN-9 addition was 16 h prior to mitoxantrone. The ability of [14C]mitoxantrone to form adducts with DNA, RNA, and protein was assessed in HL60 cells, and DNA was found to be the major substrate for adduct formation. RNA was also shown to be a good substrate while protein adduct levels were consistently very low. In mitoxantrone-resistant HL60/MX2 cells, DNA adduct levels were approximately fourfold lower. To establish the influence of DNA methylation on the ability of mitoxantrone to form adducts in cells, decitabine was used to reduce DNA methylation levels in cells prior to mitoxantrone treatment. This was clearly shown to influence adduct formation, with increasing decitabine levels leading to a decrease in the level of adducts observed in both IMR-32 and MCF-7 cell lines. Collectively, these results suggest that two major factors that influence the extent of mitoxantrone adduct formation in cells are the availability of formaldehyde and the extent of genomic DNA methylation.     

Formation and removal of B[a]P diol epoxide -- DNA adducts in human fibroblasts

Using xeroderma pigmentosum fibroblasts, deficient in excisionrepair, as controls to measure the initial rate of (±)7ß,8-dihydroxy-9,10-epoxy-7,8,9,10-tetrahydrobenzo[a]-pyrene (B[a]PDE)— DNA adduct removal in normal human fibroblasts, it wasfound that the maximum amount of carcinogen DNA adducts occurred1 h after the addition of B[a]PDE, and that during the firsthour 12% of the DNA — carcinogen adducts had already beenremoved. Thus the formation and removal of DNA — carcinogenadducts occurred simultaneously within the first hour afterB[a]PDE addition to confluent fibroblasts. Examination of excisionrepair over an extended period showed that during a further6 h, DNA adducts were removed at a rate four times slower thanthat observed during the first hour. Since the maximum levelof B[a]PDE — DNA adducts was observed 1 h after the additionof B[a]PDE to the cells in culture, this suggested that therate of breakdown of B[a]PDE was much slower than that observedin vitro. Further experiments indeed indicated that the rateof hydrolysis of B[a]PDE within the cell was significantly decreased.Thus, the stability of B[a]PDE inside the cell is governed byvery different parameters than those observed in vitro.     

Formation of cyclic deoxyguanosine adducts in Chinese hamster ovary cells by acrolein and crotonaldehyde

Acrolein and crotonaldehyde are alpha,beta-unsaturated carbonyl compounds that form 1,N2-propanodeoxyguanosine adducts when reacted with DNA in vitro. These compounds are mutagenic in Salmonella, and crotonaldehyde is tumorigenic in rats. This study used immunoassay and 32P-postlabeling methods to determine if acrolein and crotonaldehyde form these adducts in cultured mammalian cells. Adduct levels were highest in Chinese hamster ovary cells exposed to acrolein (1 mM) with 162 mumol adduct/mol deoxyguanosine. Crotonaldehyde (10 mM) formed adduct at a level of 75 mumol/mol deoxyguanosine. 32P-Postlabeling analysis confirmed the presence of adducts in crotonaldehyde-treated cells. Persistence studies showed that adduct levels were unchanged if the cells were cultured for 6 h before DNA isolation. Mutagenicity studies were performed to determine the biological consequences of these adducts. Mutations were not observed due to the toxicity of the compounds.     

Mutations induced by aminofluorene-DNA adducts during replication in human cells

To gain insight into the mechanisms by which carcinogens induce mutations in human cells, we treated a shuttle vector, pZ189, carrying the supF gene as the target for mutations with N-acetoxy-N-trifluoroacetyl-2-aminofluorene (N-AcO-TFA-AF). The plasmids were allowed to replicate in human cell line 293, and the progeny plasmids were examined for the frequency and kinds of mutations induced in supF, as well as their specific location in the sequence of the supF gene. The plasmids were reacted with N-AcO-TFA-AF so as to obtain the deacetylated adduct N-(deoxyguanosin-8-yl)-2-aminofluorene (dG-C8-AF), the principal adduct formed in DNA when mammalian cells are exposed to reactive derivatives of 2-acetylaminofluorene (AAF), including N-acetoxy-2-acetylaminofluorene. The results showed there was a linear relationship between the number of dG-C8-AF adducts per plasmid and the frequency of supF mutants induced. DNA sequencing of 47 independent mutants obtained from doses of N-AcO-TFA-AF that increased the frequency of mutants 9-15 times the background frequency and three independent mutants from lower doses showed that 92% contained point mutations, i.e. changes affecting one, or two, or three nearby bases, and that all of these point mutations involved G.C base pairs. Ninety eight percent of the point mutations were base substitutions, predominantly G.C----T.A transversions. 46% of these mutations occurred at four out of the 85 bp in the target gene (hot spots). The most prominent mutation hot spot was also the most prominent hot spot for adduct formation as judged by the frequency of termination of in vitro polymerization by the Klenow fragment on N-AcO-TFA-AF-treated plasmids.     

Formation of DNA adducts in mouse skin treated with metabolites of chrysene

Analysis by 32P-postlabelling of DNA isolated from mouse skin that had been treated in vivo with the polycyclic hydrocarbon chrysene revealed the presence of 7 adducts. All 7 adducts were also present in DNA from mice treated with trans-1,2-dihydro-1,2-dihydroxychrysene (chrysene-1,2-diol), and one of them, adduct 2, was formed from the triol derivative 9-hydroxy-trans-1,2- dihydro-1,2-dihydroxychrysene (9-hydroxychrysene-1,2-diol) and from 3-hydroxychrysene. Adducts were not detected in DNA from mice treated with trans-3,4-dihydro-3,4-dihydroxychrysene (chrysine-3,4-diol) or with 1-, 2-, 4-, 5- or 6-hydroxychrysene. In vitro modification of DNA by the anti-isomer of the bay-region diol-epoxide yielded adducts 3-7, while the corresponding triol-epoxide yielded adducts 2. It is concluded that chrysene activation in mouse skin proceeds principally via the bay-region diol-epoxide and to a lesser extent via the related bay-region triol-epoxide.     

Immunohistochemical detection of malondialdehyde-DNA adducts in human oral mucosa cells.

Malondialdehyde (MDA) is a major lipid peroxidation product that is mutagenic and tumorigenic. MDA-modified DNA adducts have been detected in animal and human tissues and may be a marker of human cancer risk. An immunohistochemical method, using a previously generated monoclonal antibody specific for MDA-DNA adducts, has been developed for the detection and quantification of DNA damage in human oral mucosa cells. The method was used initially on woodchuck liver cells treated with and without MDA, and then applied to the detection of adducts in oral mucosa cells of smokers and non-smokers. Levels of DNA damage were elevated in 25 smokers (mean relative staining intensity 97 +/- 41) compared with 25 age-, race- and sex-matched non-smokers (74 +/- 17, P < 0.02). These results demonstrate that MDA-DNA adducts can be measured in single cells of human samples by an immunohistochemical method. This methodology provides a simple way to monitor MDA-DNA damage and should be useful for studies investigating the role of exogenous and endogenous agents in oxidative stress and carcinogenesis.     

Direct determination of polyamines in human serum by radioimmunoassay.

Antipolyamine antibodies were produced following immunization of New Zealand white rabbits with thyroglobulin spermine conjugate, using multiple-site injection and macrophage-harvesting techniques. The antispermine antibody in a radioimmunoassay system cross-reacted with spermidine (22%), putrescine (1%), diaminopropane (6%), and cadaverine (0.16%). No reaction with L-lysine, ornithine, or histamine was noted. Using this antibody, the radioimmunoassay for direct determination of serum polyamines was developed. A sensitivity of 200 pg was achieved on human serum samples. This radioimmunoassay would appear to be a useful tool for the detection and monitoring of a variety of pathophysiological states associated with abnormal polyamine metabolism, i.e., cancer.     

Formation of DNA adducts in mouse tissues after intratracheal instillation of 1-nitropyrene

1-Nitropyrene (1-NP), a ubiquitous environmental pollutant,is a mammalian mutagen and causes cancer in animals. The abilityof the lung, liver and kidney to form 1-NP-DNA adducts was determinedin adult male B6C3F₁ mice following a single intratracheal instillationof 1-NP. 1-NP-DNA adducts were isolated and characterized inmouse lung, liver and kidney by HPLC analysis of the enzymaticallydigested DNA. Multiple DNA adducts were present in lung, liverand kidney at 1 day after administration. One of the major adductsin lung (20% of the total eluted radioactivity) coeluted withthe synthetic marker, N-(deoxyguanosin-8-yl)-1-amino-pyrene(C8-dG-AP). This adduct (10% of total eluted radio-activity)and others were still present in the lung at 28 days after administrationof 1-NP. One of the adducts in liver and kidney DNA digestsalso coeluted with C8-dG-AP. Treatment of the adducts with 0.3M NaOH resulting in earlier eluting peaks containing radioactivity,indicative of an imidazole ring-opening adduct. A portion ofthe original peak of radioactivity that coeluted with C8-dG-APand other adducts, however, was not affected by 0.3 M NaOH.Thus, the chromatographic properties and chemical behavior ofthe adducts formed in vivo suggest that one of the adducts inthe lung is C8-dG-AP which is formed by nitroreduction of 1-NP.Other adducts may be formed via ring-oxidation followed in someinstances by nitroreduction. These data indicate that DNA adductsof 1-NP metabolites may be formed in the lung (a primary sitefor inhaled particles), liver and kidney following inhalationof airborne particles containing 1-NP.     

Formation and removal of 7,12-dimethylbenz[a]anthracene--nucleic acid adducts in rat mammary epithelial cells with different susceptibility to carcinogenesis

The susceptibility of the rat mammary gland to 7,12-dimethylbenz[a]anthracene(DMBA)-induced carcinogenesis is maximal when DMBA is fed toyoung virgin rats, diminishing with increasing age and becomingalmost nil when pregnancy and lactation occur prior to carcinogenadministration. This present study was carried out to determinewhether these differences in susceptibility to DMBA are relatedto the ability of the gland to form and/or remove DMBA—nucleicacid adducts. Primary mammary epithelial cell cultures derivedfrom young virgin (YV), old virgin (OV) and parous (P) Sprague-Dawleyrats were treated in vitro with [³H]-DMBA for 24 h. Hydrolysatesof DNA isolated from these cells were chromatographed on SephadexLH-20 columns. In cells from the three groups of animals, thechromatographic profiles were qualitatively similar with three3H-labeled products eluting in the region expected for hydrocarbon— nucleoside adducts. This similarity in profiles suggestedthat identical DMBA—nucleic acid adducts are generatedin each group. The chromatographic data are also consistentwith the hypothesis that the hydrocarbon — DNA adductsof DMBA were generated through reactions of a "bay-region" diol-epoxideof DMBA with DNA. Excision of DMBA—DNA adducts was investigatedby measuring the time-dependent decrease in specific activityof the less dense parental DNA peak following centrifugationon alkaline CsCI gradients. Within 48 h post-DMBA treatment,P cells removed 36% of the adducts from DNA while YV and OVcells removed only 24% and 12% of the adducts respectively.In addition, P cells excised the adducts at a rate 1.5 and 5times faster than either YV or OV cells. These results suggestthat the lower susceptibility of the parous rat towards DMBA-inducedcarcinogenesis may be due not to the generation of differentDMBA — DNA adducts, but to a more efficient DNA repairmechanism that is the result of increased gland differentiationthrough the process of pregnancy and lactation.     

Formation and removal of DNA cross-links induced by melphalan and nitrogen mustard in relation to drug-induced cytotoxicity in human melanoma cells

The formation and removal of nitrogen mustard (HN2)- and melphalan-induced DNA cross-links (DNA interstrand and DNA-protein cross-links) in a human melanoma cell line (RPMI 8322), as determined by alkaline elution of DNA, was compared and related to the cytotoxic effect of each drug. HN2 was considerably more cytotoxic than melphalan as determined by inhibition of colony formation. Immediately following exposure to HN2 maximum levels of DNA cross-links were found. Melphalan, in contrast, caused a protracted induction of DNA cross-links with maximum levels obtained 6-12 h following drug exposure. HN2 induced approximately 13 times higher peak levels of DNA cross-links compared to equal concentrations of melphalan. Removal of DNA cross-links following exposure to both drugs followed an exponential time course. The rate of removal of HN2-induced DNA cross-links was, however, 1.5-2.4 times more rapid than that of melphalan-induced cross-links. A strong correlation was obtained between the cytotoxicity of both drugs and the total area under the curve for DNA interstrand cross-links, indicating that both the initial induction of as well as the rate of removal of DNA interstrand cross-links are important for the cytotoxic effects of bifunctional alkylating agents.     

Aflatoxin B; specific antibodies and their use in radioimmunoassay.

New Zealand White rabbits immunized with covalent conjugates prepared from polylysine and the O-carboxy-methyloxime derivatives of either aflatoxin B1 (AFB1) or an analogue, 5,7-dimethoxycyclopentenon (2,3-c) coumarin, produced antibodies that bind 3H-AFB1. The specificities of the antisera with respect to aflatoxins BI, B2a, G1, G2, Q1, P1, and some other structually related compounds were determined. Radioimmunoassays that can detect levels as low as 0.27 pmoles (0.06 ng) of AFB1-were used to analyze serum, urine, and crude extracts of corn and peanut supplemented with aflatoxin. In the foodstuffs, as little as 1 mug AFB1/kg was measured. The immunoassay was at least as sensitive and specific as other available analytic methods, but did not require the purification of samples by chromatography before analysis. The technique may be particularly useful in epidemiologic studies designed to study the possible relationship between chronic aflatoxin ingestion and cancer.     

Production of hybrid cells by fusion of human malignant tumour cells and primary mouse embryo cells.

Hybrid cells with a subtetraploid mouse chromosome complement were produced by fusion of three types of human tumour cells with primary mouse embryo cells. The most frequently present presumptive human chromosome was 21. Numerous chromosome rearrangements were present. Some hybrid cells produced regressing tumours in mice.     

A model for the formation and removal of hemoglobin adducts.

Hemoglobin adducts formed by chemical carcinogens can be used as biomarkers of exposure. The kinetics of adduct formation and removal is complex and depends on the processes involved in erythrocyte removal, adduct stability, and the duration and extent of exposure. In order to relate the formation of adducts to the extent of exposure in complex exposure scenarios, a model has been developed to describe the kinetics of accumulation and removal of adducts formed in vivo. The exposure scenario, lifetime of erythrocytes, and extent of adduct formation following a single exposure are required input parameters. Predictions of adduct accumulation have been generated for a wide variety of exposure scenarios and compared with both the solutions to equations derived for adduct formation and removal and experimental observations. Loss of adduct by removal of erythrocytes from circulation, both by senescence and random removal and as a result of chemical instability, has been simulated. Equations have been derived to describe the removal of hemoglobin adducts under conditions of exposure for less than the lifetime of the erythrocyte, when removal is initially a linear function of time. This model makes possible the comparison of data obtained from different exposure scenarios and in different species.