phosphorus-radioisotopes and 2-butenal

The tumorigenicity of chloral hydrate (CH), trichloroacetic acid (TCA), trichloroethanol (TCE), malondialdehyde (MDA), crotonaldehyde, acrolein, and 4-hydroxy-2-nonenal (HNE) was tested in the B6C3F(1) neonatal mouse. Mice were administered i.p. injections of CH (1000, 2000, 2500, and 5000 nmol per animal), TCA (1000 and 2000 nmol), TCE (1000 and 2000 nmol), MDA (1500 and 3000 nmol), crotonaldehyde (1500 and 3000 nmol), acrolein (75 and 150 nmol), and HNE (750 and 1500 nmol) at 8 and 15 days of age. At 12 months, only male mice treated with the positive control chemicals, 4-aminobiphenyl (500 and 1000 nmol) and benzo[a]pyrene (150 and 300 nmol), had incidences of tumors in the liver significantly higher than the solvent control. Additional male mice were dosed as described above and their livers were excised at 24, 48 h, and 7 days after the final dose. Liver DNA was isolated and analyzed by 32P-postlabeling/high-performance liquid chromatography (HPLC) and HPLC/electrochemical detection for MDA-derived adduct (M(1)G) and 8-oxo-2'-deoxyguanosine (8-OHdG) formation, respectively. At 24 and 48 h after the final dose, CH- and TCA-treated mice exhibited significantly higher M(1)G levels than the controls. 8-OHdG formation was also induced by CH, TCA, and MDA. These results suggest that under these experimental conditions the B6C3F(1) neonatal mouse is not sensitive to carcinogens that induce an increase in endogenous DNA adduct formation through lipid peroxidation or oxidative stress.

Topics: Acrolein; Aldehydes; Animals; Animals, Newborn; Carcinogenicity Tests; Carcinogens; Chloral Hydrate; Chromatography, High Pressure Liquid; Crosses, Genetic; DNA; DNA Adducts; Electrochemistry; Ethylene Chlorohydrin; Female; Lipid Peroxidation; Liver; Liver Neoplasms, Experimental; Male; Malondialdehyde; Mice; Mice, Inbred C3H; Mice, Inbred C57BL; Microsomes, Liver; Phosphorus Radioisotopes; Trichloroacetic Acid

Crotonaldehyde is an important industrial chemical to which humans and animals are ubiquitously exposed. The main intake occurs via food, tobacco smoke and possibly also via beverages. Estimation of intake via the different routes is difficult since the data available on exposure are inconsistent. Crotonaldehyde is genotoxic, mutagenic and carcinogenic and forms 1,N(2)-propanodeoxyguanosine adducts as the main DNA adducts. We have developed a (32)P-post-labeling method for these adducts based on nuclease P1 enrichment and polyethyleneimine-cellulose TLC which allows reliable detection with a detection limit of 3 adducts/10(9) nucleotides, a labeling efficiency of 80-90% and a recovery of 38%. Using this method we found crotonaldehyde adducts in different organs of Fischer 344 rats after a single gavage of high doses of 300 and 200 mg/kg body wt in the range 0.3-3.2 +/- 0.4 adducts/10(8) nucleotides and after repeated gavage of low doses of 10 and 1 mg/kg body wt (five times a week for 6 weeks) 6.2 +/- 0.2 and 2.0 +/- 0.4 adducts/10(8)nucleotides, but not in untreated animals nor in calf thymus DNA not treated with crotonaldehyde. In contrast to our results, Chung and co-workers found adducts in tissue of untreated Fischer 344 rats. This discrepancy could depend on the different methods used but also on differences in exposure of the animals via food or due to animal housing, etc.

Topics: Aldehydes; Animals; Cattle; Deoxyguanosine; DNA Adducts; Female; Phosphorus Radioisotopes; Rats; Rats, Inbred F344; Reference Standards; Sensitivity and Specificity

Crotonaldehyde is a genotoxic, mutagenic and carcinogenic alpha,beta-unsaturated carbonyl compound which forms 1,N2-propanodeoxyguanosine adducts. Humans are exposed to this compound at work places, and from tobacco smoke and air pollution, but also from food and beverages. Therefore crotonaldehyde can play a significant role in carcinogenesis. Since in vivo measurement of DNA adducts of crotonaldehyde can improve cancer risk assessment and contribute to the clarification of the role of crotonaldehyde in carcinogenicity, we developed, adapted and optimized a 32P-postlabelling technique for the adducts of crotonaldehyde based on nuclease P1 enrichment and on a polyethylene imine modified cellulose TLC to provide a detection sensitivity of three adducts per 10(9) nucleotides and a labelling efficiency of 80-90%. We also report a readily performable synthesis of adduct standards and demonstrated that DNA is completely digested to the 3'-monophosphate nucleotides under the conditions of our enzymatic DNA hydrolysis. We showed that the postlabelling method developed is appropriate for in vivo DNA-binding studies. Female Fischer 344 rats were treated by gavage with crotonaldehyde at doses of 200 and 300 mg/kg body weight, and 20 h after treatment adduct levels of 2.9 and 3.4 adducts per 10(8) nucleotides, respectively, were found in the liver DNA. Only 1.6 nucleotides per 10(8) nucleotides were found 12 h after treatment at 200 mg/kg body weight. Absolutely no adducts could be found in liver DNA of untreated rats with our method at the detection limit of three adducts per 10(9) nucleotides. In contrast to our group, the group of Chung have reported crotonaldehyde adduct levels in the range of 2.2 22 adducts per 10(8) nucleotides in DNA of untreated Fischer 344 rats. The clarification of this discrepancy is of importance for the elucidation of the role of crotonaldehyde in carcinogenicity, and both groups have decided to clarify this in cooperation in the near future.

Topics: Aldehydes; Animals; Cattle; Chromatography, High Pressure Liquid; Chromatography, Thin Layer; Deoxyguanosine; DNA Adducts; DNA Damage; Environmental Pollutants; Female; Liver; Phosphorus Radioisotopes; Rats; Rats, Inbred F344; Single-Strand Specific DNA and RNA Endonucleases

Abundant complex DNA adducts can be detected in human tissues by a combined 32P-postlabelling and high-performance liquid chromatography (HPLC) method. The HPLC profiles reveal a panorama of nuclease P1-resistant human adducts, which are not among the known human DNA adducts and are suspected of being endogenous. Lipid peroxidation-induced DNA adducts and I-compounds are two possible candidates for these adducts. Therefore, we performed two experiments: one was to identify chromatographically the lipid peroxidation-induced adducts among other human adducts with two acrolein- and crotonaldehyde-derived propano adduct standards (Acr-dG3 and Cro-dG1&2) and a structurally unknown adduct (Cro-DNA) derived from crotonaldehyde-treated DNA; and the other was to analyse the adducts in breast tissue from patients with breast cancer and from controls and to compare their behaviour with that of I-compounds in cancerous tissues. In the first experiment, Acr-dG3 and Cro-dG1 were detected in three human lung tissues, at levels ranging from 3.4 to 8.9 (x 10(-8)) and from not detectable to 2.9 (x 10(-8)), respectively. Acr-dG3 and Cro-DNA were detected in three human colon tissues, at levels of 0.2-0.4 (x 10(-8)) and 1.2-3.4 (x 10(-8)), respectively. In the second experiment, adjacent and tumorous breast tissues from 15 patients with breast cancer (of an average age of 33.4 years) and normal breast tissue from 18 controls (of an average age of 57.3) were analysed for the abundant complex adducts. The total adduct levels in the adjacent and tumorous tissues were lower than in the normal tissues (with medians of 8.0, 11.8 and 13.3 (x 10(-7)), respectively). Significant differences in the adduct levels between adjacent or tumorous tissues and normal tissues were observed in three HPLC peaks, and age was significantly associated with three peaks. These results are consistent with our speculation that the abundant adducts are comprised of lipid peroxidation-induced adducts and human homologues of I-compounds.

Topics: Acrolein; Adult; Age Factors; Aged; Aldehydes; Animals; Breast; Breast Neoplasms; Chromatography, High Pressure Liquid; Colon; DNA Adducts; Female; Humans; Lung; Male; Middle Aged; Phosphorus Radioisotopes; Rats; Tissue Distribution

Topics: Adenosine Triphosphate; Administration, Oral; Aldehydes; Animals; Carcinogens; Chromatography, High Pressure Liquid; DNA Adducts; Female; Intestine, Large; Kidney; Kinetics; Liver; Lung; Phosphorus Radioisotopes; Radioisotope Dilution Technique; Rats; Rats, Inbred F344; Tissue Distribution

Our previous study (R.G. Nath and F-L. Chung; Proc. Natl. Acad. Sci. USA, 91: 7491-7495, 1994), using a 32P postlabeling method combined with high-performance liquid chromatography specifically developed for exocyclic adducts, has shown that acrolein- and crotonaldehyde-derived 1,N2-propanodeoxyguanosine adducts (AdG and CdG, respectively) are present in the liver DNA from humans and rodents without carcinogen treatment. Those findings raised important questions regarding their role as potential endogenous DNA lesions in carcinogenesis. In this study, using a similar assay, we examined a variety of tissues from untreated rats and mice (lung, kidney, brain, breast, prostate, colon, skin, and leukocytes) and detected AdG and CdG in the DNA of these tissues. More significantly, we also obtained evidence for the presence of these adducts in the DNA of human leukocytes and mammary glands. The identities of these adducts were verified by comigration of 3', 5' -bisphosphates of the 32P-labeled adduct from DNA with the synthetic standards in a reversed-phased high-performance liquid chromatography. Additional proof of identities was provided by enzymatic conversion of AdG and CdG 3',5' -bisphosphates to the corresponding 5'-monophosphates, followed by comigration with their synthetic standards. The estimated ranges of total AdG and CdG modifications in DNA of various tissues were from 0.10 to 1.60 mumol/mol guanine for humans, based on the recoveries of external standards. This study demonstrated the ubiquity of these adducts in various tissues, suggesting their potential role as endogeneous DNA lesions in rodents and humans.

Topics: Acrolein; Adult; Aldehydes; Animals; Carcinogens; Chromatography, High Pressure Liquid; Deoxyguanosine; DNA; DNA Adducts; DNA Damage; Female; Humans; Male; Mice; Mice, Inbred A; Middle Aged; Phosphorus Radioisotopes; Rats; Rats, Inbred F344

Crotonaldehyde is mutagenic and carcinogenic and it is ubiquitous in our environment. The data base does, however, not allow an assessment of the carcinogenic risk. We have developed a sensitive 32P-postlabelling technique which allows the detection of specific DNA-adducts in animal tissues as markers for initiation of cancer cells. Adducts were found in several organs of F 344 rats after gavage and persisted to a certain extent. The determination of adduct levels in animal tissues after different exposure or even in human tissues can therefore be considered as an effect monitoring and would certainly improve the risk assessment.

Topics: Air Pollutants; Aldehydes; Animals; Biomarkers, Tumor; Carcinogens; DNA Adducts; DNA, Neoplasm; Environmental Pollutants; Food; Mutagens; Phosphorus Radioisotopes; Rats; Rats, Inbred F344; Risk Assessment; Water Pollutants

A 32P-postlabeling method is described that specifically detects and quantifies the 1,N2-propanodeoxyguanosine adducts derived from acrolein (AdG) and crotonaldehyde (CdG) and 1,N2-ethenodeoxyguanosine (EdG) in DNA. These exocyclic adducts are potential DNA lesions caused by exposure to enals as environmental pollutants and as endogenous compounds. This method was developed with the use of the synthetic adduct standards of these exocyclic adducts. The assay relies on HPLC for adduct enrichment prior to labeling and for quantitation and identification after labeling. The labeling efficiencies of adducts at the 1 fmol level ranged from 74 to 96%, whereas they were only 49-60% at the 100 fmol level. This method can detect as low as 0.2 fmol of adduct and allows the detection and quantitative determination of stereoisomers of AdG and CdG. The method was validated by using a sample of enzyme digests of 180 micrograms calf thymus DNA spiked with 25 or 75 fmol of adducts, which is equivalent to 5 or 15 adducts in 10(8) nucleotides. The recovery rates of these adducts in DNA ranged from 30 to 90% at the 25 fmol level and 21 to 55% at the 75 fmol level. Similar to the labeling efficiency, a greater recovery was observed with a lower amount of adduct in DNA. Overall, this method allows the simultaneous identification and quantification of exocyclic adducts AdG, CdG and EdG in DNA. Therefore, it provides a potential tool for studies of the in vivo formation of exocyclic adducts.

Topics: Acrolein; Aldehydes; Deoxyguanosine; DNA; Isotope Labeling; Phosphorus Radioisotopes; Stereoisomerism

People are constantly being exposed to toxic and carcinogenic aldehydes. However, little is actually known about the mechanisms underlying the toxic and carcinogenic effects of these aldehydes on human cells. The DNA alkylating activities of two of the more toxic and environmentally prominent alpha,beta-unsaturated aldehydes, acrolein and crotonaldehyde, have been studied utilizing 32P-postlabeling and nucleotide chromatographic techniques. Several putative adducts were observed in DNAs isolated from acrolein- and crotonaldehyde-treated human fibroblasts. One of these acrolein-DNA adducts was tentatively identified as the cyclic 1,N2-hydroxypropanodeoxyguanosine product, 3-(2'-deoxyribosyl)-5,6,7,8-tetrahydro-8-hydroxypyrimido[1,2- a]purine-10-one, by co-chromatography with a chemical standard. The 1,N2-hydroxypropanodeoxyguanosine along with other possible adducts, was also found in DNA isolated from peripheral blood lymphocytes obtained from a dog 1 h after receiving a therapeutic dose of 6.6 mg/kg of cyclophosphamide. These results not only demonstrate the presence of acrolein and crotonaldehyde DNA adducts in treated human cells, but also suggest that these sensitive techniques may be useful to the study of the importance of acrolein to both the carcinogenic and antineoplastic activities of cyclophosphamide and other oxazaphosphorine mustards.

Topics: Acrolein; Aldehydes; Animals; Cells, Cultured; Chromatography, Thin Layer; Cyclophosphamide; Deoxyribonucleotides; DNA; Dogs; Dose-Response Relationship, Drug; Humans; Nucleotides; Phosphorus Radioisotopes

Cyclic 1,N2-propanodeoxyguanosine adducts are formed in vitro in DNA treated with alpha-acetoxy-N-nitrosopyrrolidine or its metabolite, crotonaldehyde. However, the in vivo formation of these cyclic adducts in DNA has not been demonstrated due to the lack of a sensitive detection method. In this study, a 32P-postlabeling method specific for the detection of 1,N2-propanodeoxyguanosine adducts was developed by using the corresponding 3'-monophosphates as standards. This method was validated by using DNA modified in vitro. It was then applied for the in vivo experiments in which hepatic DNA of rats treated with N-nitosopyrrolidine (NPYR) (total dose, 1.0 mmol) in drinking water or skin DNA of Sencar mice treated topically with crotonaldehyde (1.4 mmol) was isolated and subjected to 32P-postlabeling analysis. 1,N2-Propanodeoxyguanosine adducts were detected in these DNA samples. The minimal levels of adducts from liver DNA and skin DNA detected were estimated to be approximately 0.06 and approximately 0.24 mumol/mol guanine respectively. Interestingly, a background adduct spot chromatographically indistinguishable from the 1,N2-cyclic adducts was observed in the liver DNA of untreated rats. However, no such background adduct was detected in skin DNA of mice. This method demonstrated for the first time the in vivo formation of the cyclic 1,N2-propanodeoxyguanosine adducts.

Topics: Adenosine Triphosphate; Aldehydes; Animals; Chromatography, High Pressure Liquid; Deoxyguanosine; DNA; DNA Damage; Female; Liver; Male; Mice; Mice, Inbred Strains; N-Nitrosopyrrolidine; Nitrosamines; Phosphorus Radioisotopes; Rats; Rats, Inbred F344; Skin