Relative sensitivity of 32P-postlabelling of DNA and the autoradiographic UDS assay in the liver of mice exposed to 2-acetylaminofluorene (2AAF)

In contrast to earlier studies conducted at lower dose levels, 2AAF is shown to induce a positive UDS response in the liver of mice dosed orally at dose levels between 500 and 1000 mg/kg. Similarly exposed mice had low levels of 2AAF-related hepatic DNA adducts at dose levels in the range 10-1000 mg/kg 2AAF, as determined by 32P-postlabelling analysis. It is concluded that the attenuated UDS response observed in the mouse liver, as compared to the rat liver, is due primarily to metabolic differences between these two species, coupled to a reduced capacity for UDS in the mouse liver for a given level of total 2AAF-related adducts per unit of DNA. These observations are compared and contrasted with identical studies conducted in the rat and reported in the preceding paper (Gallagher et al., 1991).     

Concomitant observations of UDS in the liver and micronuclei in the bone marrow of rats exposed to cyclophosphamide or 2-acetylaminofluorene

Oral dosing of between 5–30 mg/kg of cyclophosphamide (CP) to Alderley Park rats induced micronuclei in the bone marrow between 12 and 36 h after dosing, but failed to induce unscheduled DNA synthesis (UDS) in the liver at similar dose levels and treatment periods. Dose levels of > 30 mg/kg were toxic to the liver. In contrast, 2-acetylaminofluorene (2AAF) induced UDS in the rat liver between 4–36 h after dosing, but gave only a weak response in the bone marrow assay at dose levels between 0.5 and 2 g/kg. Selected observations were made for each chemical using both tissues of the same test animal.

It is concluded that an assessment of the genotoxicity in vivo of chemicals defined as genotoxic in vitro will contribute to an assessment of their possible mammalian carcinogenicity, and that these should involve assays conducted using both the bone marrow and the liver of rodents. Due to its relative ease of commission, the bone marrow micronucleus assay will usually be conducted first; in the case of negative results it is recommended that a liver genotoxicity assay should also be conducted. The case for employing in vivo short-term genotoxicity tests to predict the possible organotropic carcinogenicity or germ cell mutagenicity of a new in vitro genotoxin is discussed.     

Interlaboratory comparison of the 32P-postlabelling assay for aromatic DNA adducts in white blood cells of iron foundry workers

Analysis by nuclease P1-enhanced 32P-postlabelling assay of DNA isolated from the white blood cells of 53 iron foundry workers was carried out independently in 3 laboratories, and the presence of aromatic DNA adducts was detected. The mean adduct levels in foundry workers varied from 9.2 +/- 23 (laboratory 3) and 12 +/- 10 (laboratory 2) to 26 +/- 43 (laboratory 1) and for the controls from 1.7 +/- 0.7 (laboratory 3) to 3.1 +/- 1.7 (laboratory 1) adducts per 10(8) nucleotides. No effect of smoking was observed in the present study. Each laboratory observed large interindividual variations of adduct levels. Good correlations were found between the results of the 32P-postlabelling assays carried out in the 3 laboratories; the correlation coefficients between laboratories 1 and 2, 1 and 3, and 2 and 3 were 0.61, 0.62, and 0.45, respectively, all being statistically highly significant (p less than 0.01). This interlaboratory comparison of the 32P-postlabelling method indicates the reproducibility of the method and its applicability in occupational exposure monitoring.     

DNA synthesis in the ventricular myocardium of young rats exposed to intermittent high altitude (IHA) hypoxia. An autoradiographic study.

Intermittent high altitude (IHA) hypoxia (7000 m) increased the wet weight of the right ventricular myocardium of 30-day-old rats after two 4 h/day exposures. During the same period the number of DNA-synthesizing nuclei of both muscle and non-muscle cell types increased proportionally. After 4 such exposures to hypoxia the number of 3H-thymidine-labelled nuclei in both cell types increased further. In addition, the number of labelled nuclei increased significantly in the yet un-enlarged left ventricle. While there was no difference in the number of DNA-synthesizing cells between the right and left ventricles in control animals, a significant increase in the number of cells involved in DNA synthesis in the right ventricle was found in both groups of animals exposed to IHA hypoxia. These results show that DNA synthesis in myonuclei of the ventricular myocardium can be stimulated in 30-day-old rats, i.e. at the very end of the weaning period.     

Recovery of bulky DNA adducts by the regular and a modified 32P-postlabelling assay; influence of the DNA-isolation method

Bulky DNA adducts are widely used as biomarkers of human exposure to complex mixtures of environmental genotoxicants including polycyclic aromatic hydrocarbons. The 32P-postlabelling method is highly sensitive for the detection of bulky DNA adducts, but its relatively low throughput poses limits to its use in large-scale molecular epidemiological studies. The objectives of this study were to compare the impact of DNA-sample preparation with a commercial DNA-isolation kit or with the classical phenol-extraction procedure on the measurement of bulky DNA adducts by 32P-postlabelling, and to increase the throughput of the 32P-postlabelling method--whilst maintaining radio-safety--by reducing the radioisotope requirement per sample. The test DNA samples were prepared from MCF-7 cells treated with benzo[a]pyrene and from human peripheral blood lymphocytes, buffy coat, and peripheral lung tissue. The modified 32P-postlabelling procedure involved an evaporation-to-dryness step after the enzymatic digestions of the DNA, and radio-labelling with a reduced amount of [γ-32P]ATP substrate in a reduced reaction volume compared with the regular method. Higher levels of DNA adducts were measured in the MCF-7 cells and in the lung-tissue samples after isolation with the kit than after solvent extraction. A seven-fold higher level of adducts was detected in the buffy-coat DNA samples isolated with the kit than with the phenol extraction procedure (p<0.001). Reduction of the amount of [γ-32P]ATP from 50 μCi to 25 μCi (>6000 Ci/mmol specific radioactivity) per sample in the modified 32P-postlabelling procedure was generally applicable without loss of adduct recovery for all test samples prepared with both DNA isolation methods. The difference between the bulky DNA-adduct levels resulting from the two DNA-isolation procedures requires further systematic investigation. The modified 32P-postlabelling procedure allows a 50% reduction of radioisotope requirement per sample, which facilitates increased throughput of the assay whilst maintaining radio-safety.     

Lack of DNA single-strand breaks in rat liver cells exposed to 4-acetylaminofluorene, in vivo and in vitro

The induction of primary DNA damage by the non-carcinogen 4-AAF was reinvestigated in liver cells by comparison with the carcinogen 2-AAF. DNA alkaline elution showed the appearance of single-strand breaks in total liver DNA of rats 4 h after gavage with 200 mg/kg of 4-AAF. The decrease in hepatocyte viability and yield observed in these livers after collagenase perfusion indicated a cytotoxic effect of 4-AAF treatment. Viable hepatocytes isolated from 4-AAF-treated rats as well as hepatocytes from normal rats treated with 4-AAF in vitro did not present DNA single-strand breaks.     

Induction of single-strand breaks and interstrand cross-links in liver DNA after the administration of 2-acetylaminofluorene and trans-4-acetylaminostilbene to rats

2-Acetylaminofluorene (AAF) or trans-4-acetylaminostilbene (AAS) was orally or intraperitoneally administered to female Wistar rats. DNA from liver cells was analyzed for single-strand breaks by the alkaline elution assay. Only borderline effects were observed with doses (100 μMol/kg) used in animal carcinogenesis experiments. Even high doses of AAF (1,000 μMol/kg) were not effective. Methyl methanesulfonate (MMS) in vivo and gamma irradiation in vitro were shown to produce dose-dependent DNA single-strand breaks (positive control). Only a marginal effect was obtained with 100 μMollkg MMS. The elution rate of DNA was increased by a factor of 34 in liver cells in vitro with 400 rad of gamma irradiation. Only a fraction of this rate could be demonstrated immediately after irradiation in vivo, and no lesions were found two hours later. This strongly indicates the rapid repair of single-strand breaks. Additional experiments showed that AAS, a nonhepatocarcinogen, produced more interstrand cross-links in the rat liver DNA than did AAF.     

Formation and removal of DNA adducts in liver of rats treated with hepatocarcinogens 2-aminofluorene or 2-acetylaminofluorene.

The level of adducts in DNA of rats treated with 2-aminofluorene (2-AF) and 2-acetylaminofluorene (2-AAF) was compared at the times from 1 h till 28 days after injection. The highest amount of DNA adducts was observed 12 h after treatment with 2-AF and 24 h after treatment with 2-AAF, and reached values of about 18 and 21 fmol per micrograms DNA, respectively. Participation of the nonacetylated form, dG-C8-AF, in the total amount of DNA adducts was only slightly greater in rats treated with 2-AF then in those treated with 2-AAF.     

Age-related induction and disappearance of carcinogen-DNA-adducts in livers of rats exposed to low levels of 2-acetylaminofluorene

It was investigated whether in vivo aging of rat liver is associated with changes in the induction and rate of disappearance of DNA damage. For this purpose 6- and 36-month-old rats were intraperitoneally injected with a single, low dose (5 mg/kg body wt.) of the model liver carcinogen 2-acetylaminofluorene (AAF). Using the 32P-postlabeling assay we found that N-(deoxyguanosin-8-yl)-2-aminofluorene (dG-C8-AF) was the major DNA-adduct formed. The minor adduct N-(deoxyguanosin-8-yl)-2-acetylaminofluorene (dG-C8-AAF) could only be detected after doses of 20 mg/kg or more. Quantitation of adduct levels at various time points after treatment indicated a rapid induction of AF-adducts, which were already present at 6 h after treatment. The subsequent loss of AF-adducts was relatively slow, as was indicated by the presence of a substantial amount of AF-adducts as late as 21 days after treatment. Slight age-related differences in the pattern of induction and disappearance of AF-adducts and a somewhat higher level of persisting lesions in old than in young rats were observed.     

Evaluation of four methods for scoring cytoplasmic grains in the in vitro unscheduled DNA synthesis (UDS) assay

The in vitro unscheduled DNA synthesis (UDS) assay measures DNA repair (incorporation of [³H]thymidine) following in vitro treatment of rat primary hepatocytes. The autoradiographic method was used to detect UDS by counting developed silver grains in the photographic emulsion overlaying nuclei and cytoplasmic areas of the hepatocytes. In this communication we report results using 4 scoring methods: (1) the most heavily labeled cytoplasmic areas adjacent to the nucleus (our standard method), (2) the cytoplasmic area left of the nucleus, (3) the cytoplasmic areas left and right of the nucleus, and (4) 2 cytoplasmic areas whose positions were selected at random. Rat primary hepatocyte cultures treated with a medium control, a solvent control (dimethyl sulfoxide) and 5 known genotoxic chemicals (2-acetylamino-fluorene, dimethylnitrosamine, diethylnitrosamine, methyl methanesulfonate and ethyl methanesulfonate) were scored using these 4 methods. The average or maximum cytoplasmic grain count was subtracted from the nuclear grain count to yield net grains/nucleus (NG). In general, NG counts for Methods 2,3 and 4 were similar, although shifted about 3–10 grains higher than Method 1 for controls and most treated groups. Methods 2, 3 and 4 showed more experiment-to-experiment variability in sensitivity for detecting statistically significant increases in treated groups than did our standard method. Thus, the alternative methods afforded no consistent improvements in sensitivity or reduction of variability for this assay. Subtraction of the average or the highest cytoplasmic count had virtually no effect on the sensitivity of the assay, but simply requires an appropriate adjustment of the criteria for a positive response.     

32P-postlabelling analysis of 1,3-butadiene-induced DNA adducts in vivo and in vitro

Butadiene monoepoxide (BMO), epoxybutanediol (EBD) and diepoxybutane (DEB) are reactive metabolites of 1,3-butadiene (BD), an important industrial chemical classified as a probable human carcinogen. The covalent interactions of these metabolites with DNA lead to the formation of DNA adducts which may induce mutations or other types of DNA damage, resulting in tumour formation. In the present study, two pairs of diastereomeric N-1-BMO-adenine adducts were identified in the reaction of BMO with 2´-deoxyadenosine-5´-monophosphate (5´-dAMP). The major products formed by reacting EBD with 2´-deoxyguanosine-5´-monophosphate (5´-dGMP) were characterized as diastereomeric N-7-(2´,3´,4´-trihydroxybut-1´-yl)-5´-dGMP by UV and electrospray mass spectrometry. The formation of N-7-BMO-guanine adducts (1´-carbon, 60; 2´carbon, 54/10⁴ nucleotides) in BMO-treated DNA was about four times higher than that of N-1-BMO-adenine adducts (1´-carbon, 20; 2´-carbon, 8.7/10⁴ nucleotides). However, the recovery of N-1-BMO-adenine adducts in DNA (45 ± 5%) was two times higher than that of N-7-guanine adducts (20 ± 4%) by 32P-postlabelling analysis. Using the 32P-postlabelling/ HPLC assay, N-1-BMO-adenine, N-7-BMO-guanine and N-7-EBDguanine adducts were detected in BMO- or DEB-treated DNA and in liver DNA of rats exposed to BD by inhalation. The amount of N-7-EBD-guanine adducts (11/10⁸ nucleotides) in rat liver was about three-fold higher than N-7-BMO-guanine adducts (4.0/10⁸ nucleotides). The novel finding of N-1-BMO-adenine adducts formed in vivo may contribute to the understanding of the mechanisms of BD carcinogenic action.     

The effects of putative DNA repair inhibitors on DNA adduct levels and unscheduled DNA synthesis in rat hepatocytes exposed to 2-acetylaminofluorene

The enzymology of DNA repair is currently under active investigation. The purpose of the present study was to examine the involvement of a number of enzymes (DNA polymerase alpha and beta, DNA topoisomerase II and ribonucleotide reductase) in the repair of chemically induced DNA damage in a mammalian cell system. This was done by studying the effects of inhibitors of these enzymes on the levels of 2-acetylaminofluorene (2-AAF)-DNA adducts and on the induction of UDS in primary cultures of rat hepatocytes exposed to the carcinogen in vitro. The results obtained with aphidicolin (an inhibitor of DNA polymerase alpha) show that the binding of 2-AAF to cellular DNA was significantly higher in samples exposed to this compound. Moreover, induction of UDS by 2-AAF was completely blocked in the presence of this compound. Dideoxythymidine, a DNA polymerase beta inhibitor, led to complex results. It produced a reduced DNA-specific activity due to [3H]2-AAF adduct formation as well as a diminished but still detectable UDS response in the presence of 2-AAF. Inhibitors of DNA topoisomerase II (nalidixic acid) and ribonucleotide reductase (hydroxyurea) did not cause any statistically significant change in the accumulation of 2-AAF adducts nor did they affect the induction of UDS. The data clearly suggest that DNA polymerase alpha participates in the repair of 2-AAF adducts in hepatocytes. In addition, neither DNA topoisomerase II activity, nor limitations in the precursor nucleotide pools appear to be critical factors in this process.     

Application of the neutral red assay (NR assay) to monolayer cultures of primary hepatocytes: rapid colorimetric viability determination for the unscheduled DNA synthesis test (UDS)

The neutral red (NR) absorption method was adapted for the determination of cell viability in the UDS assay with primary hepatocyte cultures of the rat. The NR method is rapid, easy to perform, and suitable for handling of large numbers of cultures simultaneously. It can be used for concentration range-finding pre-experiments. In addition, it can easily be integrated into a UDS test protocol for documentation of toxic effects if supplementary cultures for each concentration are established. The time schedule required for the NR assay makes it possible for one person to process the hepatocytes for autoradiography and at the same time determine the toxicity.     

Effect of N-2-acetylaminofluorene on poly(ADP-ribose) polymerase activity and DNA synthesis in cultured rat hepatocytes exposed to epidermal growth factor.

The nuclear enzyme poly(ADP-ribose) polymerase is involved in basic cellular processes such as DNA replication and repair, cell differentiation and transformation, gene expression. We have studied the effect of 2AAF, a genotoxic aromatic amine, on pADPRP activity during DNA synthesis stimulated by EGF, using the cultured rat hepatocytes model. DNA synthesis was measured as [3H]thymidine incorporated/microgram DNA while pADPRP activity was expressed in pmol[32P]NAD incorporated/min/microgram DNA. Our results show that 2AAF treatment of EGF-stimulated rat hepatocytes induces a full block of DNA replication which is preceded and accompanied by a net inhibition of endogenous and total pADPRP activity, respectively. A block in pADPRP activity in normal hepatocytes, exposed to 2AAF in vitro or in vivo, could play a key role in cell transformation. Our data add further information on the possible involvement of this nuclear catalytic activity during DNA replication.     

The relationship between aryl hydrocarbon hydroxylase activity and DNA adducts measured by 32P-postlabelling assay in lymphocytes of lung cancer patients

We have investigated the correlation between DNA adduct levels and aryl hydrocarbon hydroxylase (AHH) activity in peripheral lymphocyte samples obtained from 42 lung cancer patients. DNA adducts and AHH activity were determined by the ³²P-postlabelling technique and the fluorometric method, respectively. The mean +/- SD of DNA adduct level was 0.88 +/- 0.37 (ranged from 0.22 to 1.90) per 10⁸ nucleotides. The geometric means of non-induced and 3-methylcholanthrene (MC)-induced AHH activity, as well as AHH inducibility (MC-induced AHH activity/non-induced AHH activity) were 0.029, 0.228 pmol min^-1 10^-6 cells, and 7.776, respectively. There was no statistically significant correlation between DNA adduct levels and non-induced or MC-induced AHH activity. A tendency of positive correlation was found between DNA adduct levels and AHH inducibility for the all subjects (n = 42, r = 0.25, p = 0.11). Such a positive correlation reached statistical significance in the subjects with squamous cell carcinoma (n = 13, r = 0.70, p &lt; 0.01). In addition, similar correlation of DNA adducts with AHH inducibility was also observed in the GSTM1 present genotype (n = 17, r = 0.44, p = 0.07) and GSTP1-AA genotype (n = 29, r = 0.37, p = 0.05) individuals. These findings suggest that DNA adduct levels are mediated by CYP1A1 enzyme, and AHH inducibility may be a more relevant indicator than specific AHH activity for explaining the variation of DNA adduct levels in lymphocytes.     

The in vitro unscheduled DNA synthesis (UDS) assay in rat primary hepatocytes: evaluation of 2-furoic acid and 7 drug candidates

The in vitro unscheduled DNA synthesis assay (UDS) is part of the routine genetic toxicology screening at The Upjohn Company. The purpose of this paper is to report results for 8 compounds which were tested in the in-house genetic toxicology program. These compounds represent diverse chemical structure and most of them entered the screening program because they are biologically active in efficacy screens. All tests were carried out under Good Laboratory Practices Regulations of the U.S. Food and Drug Administration. None of the materials reported here produced an increase in UDS and therefore the UDS results with these compounds do not suggest potential for genotoxicity.     

A liver micronucleus assay using young rats exposed to diethylnitrosamine: methodological establishment and evaluation

We have developed a simple liver micronucleus assay using young rats (up to 4 weeks old) to assess cytogenetic damage of chemicals in liver cells. Diethylnitrosamine (DEN) was used as a model rodent hepatocarcinogen in this study. Compared to the partial hepatectomy method most commonly used for the liver micronucleus assay, the present assay method showed equal or even higher practicability. The young rat liver micronucleus assay was also characterized for rat strain differences, sampling time after treatment, single treatment vs. split treatment, age of animals, administration route, and staining method. Although based on one model chemical, we propose an acceptable protocol for the micronucleus assay using young rat liver as follows: Up to 4-week-old rats should be used; oral or intraperitoneal administration can be used; single or repeated treatment protocols can be applied; sampling time is 3-5 days after the last treatment; hepatocytes are prepared by the collagenase perfusion method; and cells are stained with the AO-DAPI double staining method.