DNA-protein cross-link formation in Burkitt lymphoma cells cultured with benzaldehyde and the sedative paraldehyde.

Exposure to aldehydes represents potential risks to human and animal health. Cyclic aldehydes such as benzaldehyde, 2-furaldehyde, and paraldehyde were found to induce formation of stable DNA-protein cross-links (DPXs) in cultured human lymphoma cells. A relationship between increased cytotoxicity and DPX formation was observed with each aldehyde. Paraldehyde is a sedative drug used predominately in treatment of ethanol withdrawal. Paraldehyde was the most potent cross-linking aldehyde studied, yet least cytotoxic. Although DPX formation by aliphatic aldehydes is well-known, this study confirms the potential for cyclic aldehydes to cause formation of DPXs in cultured cells at therapeutically relevant doses.

Critical factors in assessing risk from exposure to nasal carcinogens.

Anatomical, physiological, biochemical and molecular factors that contribute to chemical-induced nasal carcinogenesis are either largely divergent between test species and humans, or we know very little of them. These factors, let alone the uncertainty associated with our knowledge gap, present a risk assessor with the formidable task of making judgments about risks to human health from exposure to chemicals that have been identified in rodent studies to be nasal carcinogens. This paper summarizes some of the critical attributes of the hazard identification and dose-response aspects of risk assessments for nasal carcinogens that must be accounted for by risk assessors in order to make informed decisions. Data on two example compounds, dimethyl sulfate and hexamethylphosphoramide, are discussed to illustrate the diversity of information that can be used to develop informed hypotheses about mode of action and decisions on appropriate dosimeters for interspecies extrapolation. Default approaches to interspecies dosimetry extrapolation are described briefly and are followed by a discussion of a generalized physiologically based pharmacokinetic model that, unlike default approaches, is flexible and capable of incorporating many of the critical species-specific factors. Recent advancements in interspecies nasal dosimetry modeling are remarkable. However, it is concluded that without the development of research programs aimed at understanding carcinogenic susceptibility factors in human and rodent nasal tissues, development of plausible modes of action will lag behind the advancements made in dosimetry modeling.

Measurement of DNA-protein cross-links in human leukocytes following acute ingestion of chromium in drinking water.

Increased DNA-protein cross-linking (DPX) in circulating leukocytes has been proposed as a potential biomarker for exposure and genotoxic damage caused by inhalation of certain reactive chemicals, such as hexavalent chromium [Cr(VI)]. This study was designed to determine whether ingestion of a single dose of potassium dichromate alone [Cr(VI)] or potassium dichromate fully reduced to Cr(III) with orange juice (prior to ingestion) causes an increase in DPX of circulating leukocytes in humans. Four adult male volunteers ingested a bolus dose of 5000 micro chromium in a 0.51 volume of water (10 p.p.m.), and blood samples were collected at 0, 60, 120, 180 and 240 min afterwards for analysis of DPX formation in circulating leukocytes. Results were compared to each person's own background concentration of DPX in leukocytes. Blood and urine samples were also collected for up to 2 weeks following the dose to examine the pattern of uptake and excretion of chromium. The results showed that there was no significant change in DPX observed following either Cr(VI) or Cr(III) ingestion, even though blood and urine chromium measurements indicated systemic uptake of a substantial fraction of the ingested chromium. Since Cr(III) does not possess DPX-inducing properties while Cr(VI) does, these results suggest that the Cr(VI) was reduced to Cr(III) intragastrically prior to absorption or that the amount of Cr(VI) absorbed into the blood was insufficient to produce DPX. These results are consistent with prior research that indicated that DPX would not occur following exposure to Cr(VI) except at very high doses.

DNA-protein crosslink formation in rat nasal epithelial cells by hexamethylphosphoramide and its correlation with formaldehyde production.

Hexamethylphosphoramide (HMPA) is an aprotic polar solvent and nasal carcinogen in rats. The metabolism of HMPA to formaldehyde, another nasal carcinogen in rats, was found to be approximately 6 times greater in microsomes from olfactory tissues than from respiratory tissues (isolated from both male and female rats). HMPA was shown to induce formation of DNA-protein crosslinks (DPXLS) in isolated rat nasal epithelial cells. Using a filter binding assay, we demonstrated that microsomal activation is necessary for HMPA-induced crosslink formation between plasmid DNA and calf thymus histones, presumably through metabolic N-demethylation of HMPA and the formation of formaldehyde. Both formaldehyde production and DPXL formation were inhibited by pre-incubation of nasal mucosal extracts with metyrapone, an inhibitor of cytochrome P-450. Significant dose-dependent increases in DPXL formation were observed in respiratory and olfactory epithelial cells exposed to > or = 0.5 and 1 mM HMPA, respectively, for 3 h at 37 degrees C. This resulted in DPXL accumulation at 18-20% higher levels than untreated cells. Increases in DPXL formation in rat nasal epithelial cells cultured with 1 mM HMPA were inhibited by over 70% by co-administration of metyrapone. These data suggest that metabolic liberation of formaldehyde from HMPA is involved in the mechanism of HMPA-induced nasal carcinogenesis. Comparative studies showed formaldehyde to be more potent than HMPA in the induction of DPXL in nasal epithelium. However, induction of tumor formation after two years at 50 ppb HMPA and 6 ppm formaldehyde show the former to be active at several-fold lower concentrations. Therefore, other mechanisms are likely to be involved in HMPA nasal carcinogenesis.

Formation and stability of acetaldehyde-induced crosslinks between poly-lysine and poly-deoxyguanosine.

The amino acid residue and nucleoside specificity of acetaldehyde-induced DNA-protein crosslinks (DPXLs) were studied using a modified filter binding assay. A 40% inhibition of acetaldehyde-induced pUC13 plasmid DNA-calf thymus histone crosslink formation was achieved by addition of 50 mM lysine (free base), while arginine was unable to affect crosslink formation at concentrations to 150 mM. Polymers (5-mers) of lysine (poly-lys5) were able to substitute for histones in acetaldehyde-induced plasmid crosslink formation, being equally effective at equimolar concentrations. Homopolymers (6-mers) of deoxyguanosine (poly-dG6) (but not deoxyadenosine, deoxycytidine or thymidine) served as an efficient substrate for acetaldehyde-induced DPXL formation, using either calf thymus histones or poly-lys5 as the protein source. Acetaldehyde-induced crosslinks between poly-dG6 and poly-lys5 were formed rapidly, but were unstable at 37 degrees C (a half-life or 1.5-2 h). Stability of these crosslinks was unaffected by pH at a range of 5.5-9.0 at 37 degrees C for 2 h. Results presented here suggest that unstable complexes of deoxyguanosine and lysine constitute a major portion of the DPXLs formed by acetaldehyde in vitro.

Cytotoxicity and DNA-protein crosslink formation in rat nasal tissues exposed to vinyl acetate are carboxylesterase-mediated.

Vinyl acetate is used in the paint, adhesive, and paper board industries. Vinyl acetate is a nasal carcinogen in rats exposed by inhalation for 2 years to 200 and 600 ppm, but not 50 ppm. Previous studies from our laboratory suggest that rat liver microsome-activated vinyl acetate induces plasmid DNA-histone crosslinks, in vitro, through esterase-mediated metabolism. Since nasal tissues contain high levels of carboxylesterase, tumorigenesis may be related to in situ production of the hydrolysis products acetaldehyde and acetic acid. Vinyl acetate was cytotoxic to both respiratory and olfactory tissues in vitro at 50-200 mM, but not 25 mM, after 2 hr exposure. Pretreatment of rats with the carboxylesterase inhibitor, bis-(p-nitrophenyl) phosphate (BNPP), attenuated the cytotoxic effects and metabolism of vinyl acetate in both tissue types. Semicarbazide, an aldehyde scavenger, was unable to protect the tissues from vinyl acetate-induced cytotoxicity. When the metabolites were tested, acetic acid, but not acetaldehyde, was cytotoxic to both tissues. The induction of DNA-protein crosslink (DPXL) formation by acetaldehyde and vinyl acetate in rat nasal epithelial tissues was detected using a sodium dodecyl sulfate/KCl precipitation technique. Endogenous crosslink levels ranged from 0.5 to 2.0% of total DNA and were considered background. Epithelial cells isolated from both olfactory and respiratory turbinates exhibited dose- and time-dependent increases in DPXL formation when exposed to 10-150 mM acetaldehyde for 1-2 hr at 37 degrees C. Similarly, respiratory and olfactory epithelial cells exposed to 5-75 mM vinyl acetate for 1-2 hr accumulated up to 12- and 15-fold higher crosslink levels than untreated cells, respectively. However, vinyl acetate appears to induce much higher levels of DPXLs at equimolar doses than acetaldehyde. This is thought to be related to stimulation of acetaldehyde-induced DPXL formation by the pH lowering effect of acetic acid production (via vinyl acetate hydrolysis). Pretreatment of the nasal turbinates with 1 mM BNPP reduced 25 mM vinyl acetate-induced DPXL formation by over 75% in both tissues. These data support a hypothesis that carboxylesterase-mediated hydrolysis of vinyl acetate is necessary to generate the active intracellular cross-linking agent, acetaldehyde, and the cytotoxic metabolite, acetic acid.

Reaction kinetics of DNA-histone crosslinking by vinyl acetate and acetaldehyde.

The formation and stability of DNA-protein crosslinks (DPXLs) formed by incubation of pUC13 plasmid DNA and calf thymus histones with 1-100 mM acetaldehyde was studied using a filter binding assay. DPXLs were formed at a rate of 127 DPXLs/plasmid molecule/mmol acetaldehyde in a reaction containing 1 microgram of histones and 0.33 microgram of DNA at 37 degrees C for 1 h. Acetaldehyde-induced DPXLs were unstable at 37 degrees C, with loss of up to 75% by 8 h. Crosslink formation was significantly higher at lower pH, with 3- and 2-fold higher levels at pH 5 and 6 respectively than at pH 7.5. Induction of DPXL formation by 1-100 mM vinyl acetate in the presence of rat liver microsomes was observed at 37 degrees C over 3 h. DPXL accumulation followed S-phase enzymatic kinetics, with a rate of formation of 1.1 DPXLs/plasmid molecule/mmol vinyl acetate/microgram microsomal protein/microgram DNA. Vinyl acetate was unable to cause formation of DPXLs in the absence of microsomes. A carboxylesterase inhibitor, bis-(p-nitrophenyl) phosphate, was able to block DPXL formation by vinyl acetate and microsomes. This work supports the hypothesis that DPXL formation by vinyl acetate requires microsomal metabolism to acetaldehyde, which is the active crosslinking agent.

Efficiency of DNA-histone crosslinking induced by saturated and unsaturated aldehydes in vitro.

Using a filter-binding assay based on precipitation of pUC13 plasmid DNA bound to calf-thymus histones, we have determined the efficiency of formation of DNA-protein crosslink formation induced by several aldehyde compounds in vitro. Formaldehyde, glutaraldehyde and acrolein were the most potent, causing 1 crosslink per 2.7 kbp of DNA at 1.5, 8 and 150 microM, respectively. All other compounds tested gave 1 crosslink per plasmid molecule in the mM concentration range as follows: acetaldehyde, 115 mM; propionaldehyde, 295 mM; butyraldehyde, 360 mM; crotonaldehyde, 8.5 mM; trans-2-pentenal, 6.3 mM. Significant decreases in the efficiency of DPXL formation were observed with monofunctional aldehydes of higher carbon chain length. For example, the concentration of formaldehyde needed to give 1 crosslink per molecule was almost 10(5) times less than that of acetaldehyde. Acetaldehyde differs from formaldehyde only by one saturated carbon. The presence of an unsaturated bond between the 2-3 carbons improved the potential for crosslink formation. For example, acrolein was over 500-fold more potent than propionaldehyde. Glutaraldehyde was almost as potent as formaldehyde, indicating that the bifunctional nature of this 5-carbon saturated aldehyde may be crucial to its high efficiency of DNA-protein crosslinking.

Rapid and quantitative separation of nicotinamide and its N1-methylated metabolite by Dowex AG50-X4 chromatography.

The use of column chromatography with Dowex AG50-X4 resin has allowed the quantitative separation of nicotinamide from its primary metabolite, N1-methylnicotinamide. Although the sensitivity is similar to earlier high-performance liquid chromatographic methods, this procedure allows multiple assays to be carried out simultaneously in a matter of minutes. This method should be useful to study nicotinamide methyltransferase activity in either whole cells or extracts, and is particularly well suited to screen column fractions for enzyme purification purposes.

2-Aminobenzamide, an inhibitor of ADP-ribosylation, antagonizes induced DNA hypomethylation during differentiation of murine Friend erythroleukemia cells by N'-methylnicotinamide.

The ADP-ribose synthesis inhibitor 2-aminobenzamide was found to reduce the induction of haemoglobin synthesis in murine Friend erythroleukemia cells, cultured continuously for 96 h with 5 mM N'-methylnicotinamide, with over 50% inhibition at equimolar doses. 2-Aminobenzamide was optimally effective as an inhibitor of differentiation if present only during the time period 24-48 h after initial N'-methylnicotinamide exposure. A transient, genome-wide DNA hypomethylation accompanies induction by N'-methylnicotinamide. Although 2-aminobenzamide does not seem to affect normal DNA methylation in cultured cells, the observed DNA hypomethylation in cells cultured for 24 h with 5 mM N'-methylnicotinamide was antagonized over 50% by equimolar 2-aminobenzamide. We suggest that ADP-ribosylation has a positive modulatory role in erythroid differentiation, and possibly in the process(es) leading to DNA hypomethylation following inducer exposure.

Inhibition of DNA methylation in Friend erythroleukemia cells by actinomycin D.

Actinomycin D caused the production of hypomethylated DNA in cultured Friend erythroleukemia cells at cell culture concentrations of 1-4 ng per ml. Inhibition of DNA methyltransferase in cell-free assays was kinetically complex, with mixed-type inhibition. Cornish-Bowden graphical analysis was used to derive a Ki of about 35 nmol Act D per mg DNA. Although nuclei from drug-treated cells were found to contain hypomethylated DNA and DNA methyltransferase could be extracted from the nuclei, the methyl-accepting ability of DNA in whole nuclei themselves was not elevated. We conclude that the low level of Act D bound to DNA in the nuclei is sufficient to prevent the remethylation of hypomethylated sites.

Detection of 5-methylcytosine removal from DNA of Friend erythroleukemia cells following exposure to several differentiating agents.

The use of a 32P-labeling procedure has allowed us to monitor the stability of 5-methylcytosine (5-mC) in the existing fraction of DNA in differentiating Friend erythroleukemia cells (FELCs). The levels of 5-mC in existing DNA of control cells remained constant for 20 h, at nearly 3.95% of total labeled cytosine. Exposure of pre-labeled cells to 5 mM N'-methylnicotinamide (N'-MN), 4 mM hexamethylene bisacetamide (HMBA), or 270 mM dimethylsulfoxide (DMSO) over the same time-period resulted in the loss of 6.1, 4.8 and 1.8%, respectively, of the 5-mC from the pool of labeled cytosine present in the existing fraction of the DNA.