Significant Increase In Unscheduled DNA Synthesis Research Articles

Potent genotoxic activity of benzo[a]pyrene coated onto hematite measured by unscheduled DNA synthesis in vivo in the rat.

Since epidemiological studies have firmly implicated co-exposure to iron oxides and polycyclic aromatic hydrocarbons as a potential etiological factor involved in the excess mortality due to lung cancer in miners, experimental studies have been performed to investigate the role of iron particles in benzo[a]pyrene (B[a]P)-induced lung pathogenesis. In a previous study using the Comet assay in vivo in the rat, we demonstrated that iron particles enhanced B[a]P genotoxicity. To determine whether co-exposure (B[a]P/iron oxides) induces a real genotoxic activity or is only due to inhibition of DNA repair, the unscheduled DNA synthesis (UDS) assay was implemented in vivo in the rat. The UDS assay was used to measure DNA repair in two cell types (lung cells and hepatocytes) of OFA Sprague-Dawley rats, 24 h after endotracheal administration of a single dose of an iron oxide (hematite, Fe2O3) (0.75 mg), of B[a]P (0.75 mg) or of B[a]P (0.75 mg) coated on hematite particles (0.75 mg). No difference in UDS was observed in the two organs investigated in rats treated with iron oxide alone compared with control animals, while a significant increase in UDS was observed in lungs and liver of rats treated with B[a]P alone compared with control animals. The main finding was a significant increase in UDS observed in both lung and liver cells of rats treated with B[a]P coated on hematite when compared with those treated with B[a]P alone. The current study demonstrates (i) that iron particles did not inhibit UDS in lung cells and hepatocytes of OFA Sprague-Dawley treated rats with B[a]P coated on hematite and (ii) a potent genotoxic activity of co-exposure to B[a]P coated on hematite. Therefore, our data may contribute to explaining the excess mortality due to lung cancer in epidemiological studies and overall why exposure to B[a]P coated on Fe2O3 particles resulted in a higher tumor incidence in rodents compared with exposure to B[a]P alone.

Lack of effect of coumarin on unscheduled DNA synthesis in the in vivo rat hepatocyte DNA repair assay

The ability of coumarin to induce UDS in male Sprague–Dawley CD rat hepatocytes in vivo was assessed using the unscheduled DNA synthesis (UDS) assay. From a preliminary toxicity study the oral maximum tolerated dose (MTD) of coumarin was determined to be 320 mg/kg body weight. For the UDS studies, rats were treated with 0 (corn oil control), 32 (one-tenth the MTD), 107 (one-third the MTD) and 320 (MTD) mg/kg coumarin via oral gavage. Rats were also treated with 20 mg/kg body weight dimethylnitrosamine (DMN) or 50 mg/kg body weight 2-acetylaminofluorene (2-AAF) as positive controls for the 2–4 hr and 12–16 hr expression of UDS, respectively. Hepatocytes were isolated by liver perfusion either 2–4 hr or 12–16 hr after treatment and cultured in medium containing [ methyl- 3H]thymidine for 4 hr and assessed for UDS by grain counting of autoradiographs. Coumarin treatment at doses of 32–320 mg/kg body weight had no statistically significant or dose-related effect on UDS in rat hepatocytes either 2–4 hr or 12–16 hr after dosing. In contrast, both DMN 2–4 hr after dosing and 2-AAF 12–16 hr after dosing produced significant increases in UDS assessed as the net nuclear grain count. Both genotoxins also increased the percentage of hepatocyte nuclei with greater than 5 net grains. Treatment with coumarin, DMN and 2-AAF had no statistically significant effect on the proportion of rat hepatocytes undergoing replicative DNA synthesis. In summary, this study demonstrates that coumarin does not induce UDS in hepatocytes of male Sprague–Dawley CD rats after oral administration at doses up to the MTD of 320 mg/kg. The responsiveness of the animals used in this study to genotoxic agents was demonstrated by the clear induction of DNA repair after treatment with DMN and 2-AAF.

Lack of Effect of Coumarin on Unscheduled DNA Synthesis in Precision-cut Human Liver Slices

In this study the effect of coumarin on unscheduled DNA synthesis (UDS) in precision-cut human liver slices has been examined. Liver slices from tissue samples from four donors were cultured for 24 hr in medium containing [ 3H]thymidine and 0–5.0 m m coumarin using a dynamic organ culture system and processed for autoradiographic evaluation of UDS. As positive controls liver slices were also cultured with three known genotoxic agents, namely 0.02 and 0.05 m m 2-acetylaminofluorene (2-AAF), 0.002 and 0.02 m m aflatoxin B 1 (AFB 1) and 0.005 and 0.05 m m 2-amino-1-methyl-6-phenylimidazo [4,5- b]pyridine (PhIP). UDS was quantified as the net grain count in centrilobular hepatocytes and as the percentage of centrilobular hepatocyte nuclei with more than five net grains. Compared with control liver slice cultures, treatment with 0.05–5.0 m m coumarin had no effect on UDS. In contrast, treatment with 0.02 and 0.05 m m 2-AAF, 0.002 and 0.02 m m AFB 1 and 0.005 and 0.05 m m PhIP produced significant increases in the net grain counts of centrilobular hepatocytes. The greatest induction of UDS was observed in liver slices treated with 0.05 m m PhIP. Treatment with 2-AAF, AFB 1 and PhIP also produced significant increases in the number of centrilobular hepatocyte nuclei with more than five net grains. At the concentrations examined neither coumarin, 2-AAF, AFB 1 nor PhIP had any significant effect on replicative DNA synthesis in 24 hr cultured human liver slices. These results demonstrate that coumarin does not induce UDS in cultured human liver slices. However, all three positive control compounds produced marked significant increases in UDS, thus confirming the functional viability of the human liver slice preparations used in this study. The results of this study suggest that coumarin is not a genotoxic agent in human liver.

Effect of Some Cooked Food Mutagens on Unscheduled DNA Synthesis in Cultured Precision-cut Rat, Mouse and Human Liver Slices

Precision-cut liver slices were prepared from male Fischer 344 rats, female CDF 1 mice and humans (both male and female subjects). Liver slices were cultured for 24 hr in medium containing [ 3H]thymidine and either PhIP, IQ, MeIQ, MeIQ x, Glu-P-1 or Trp-P-1, and then processed for autoradiographic evaluation of unscheduled DNA synthesis (UDS). All six cooked food mutagens examined produced concentration-dependent increases in UDS in human liver slices. PhIP was the most potent compound examined, followed by MeIQ x, IQ and then MeIQ, Glu-P-1 and Trp-P-1. Significant increases in UDS were observed with PhIP, IQ and MeIQ x at concentrations as low as 5 μ m in the culture medium. The same rank order of potency was not apparent in either rat or mouse liver slices. In rat liver slices only MeIQ significantly induced UDS, although positive results were obtained with two other genotoxins, namely 2-acetylaminofluorene and aflatoxin B 1. Apart from MeIQ x, all the cooked food mutagens produced significant increases in UDS in mouse liver slices. This study demonstrates the usefulness of precision-cut liver slices to evaluate species differences in xenobiotic-induced genotoxicity. Both marked compound and species differences in induction of UDS were observed. The data provide further evidence that dietary cooked food mutagens are potential human carcinogens.

Production of unscheduled DNA synthesis in rodent hepatocytes in vitro, but not in vivo, by 3-chloro-4-(dichloromethyl)-5-hydroxy-2[5 H]-furanone (MX)

Incubation of both rat and mouse hepatocytes with 3-chloro-4-(dichloromethyl)-5-hydroxy-2[5 H]-furanone (MX) in vitro resulted in a dose-dependent increase in unscheduled DNA synthesis (UDS) at sub-cytotoxic concentrations (1–10 μM MX; 20 h incubation). Depletion of glutathione stores by pre-treatment of rat hepatocytes with buthionine sulfoximine did not result in a significant increase in UDS produced by MX. In contrast, MX did not induce UDS in mouse hepatocytes ex vivo either 3 or 16 h following administration of a single oral dose of 100 mg/kg MX. Despite the ability of MX to produce repairable DNA damage, restricted access of MX to the liver may prevent a measurable UDS response in vivo.

Genotoxicity of tacrine in primary hepatocytes isolated from B6C3F1 mice and aged ad libitum and calorie restricted Fischer 344 rats

Tacrine (1,2,3,4-tetrahydro-9-aminoacridine; THA), a reversible centrally acting anticholinesterase, has been shown to be potentially useful for treatment of patients with Alzheimer's disease. However, currently available forms of THA may be therapeutically limited by the fact that high doses have resulted in liver and kidney damage. To determine if THA is hepatotoxic via a genotoxic mechanism, we evaluated its ability to induce unscheduled DNA synthesis (UDS) in primary cultures of rodent hepatocytes. Positive dose-dependent increases in UDS were observed in hepatocytes derived from male B 6C 3F 1 mice and from young, middle-aged, and old Aroclor-induced (ARO) male F344 rats maintained on either an ad libitum (AL) or a caloric restricted (CR) diet (60% of AL) and exposed to 0.05–1000.0 μg/ml of THA. Hepatocytes from old AL rats, treated with THA, exhibited significant age-related decreases in DNA repair compared to young and middle-aged AL rats. By contrast, cultures from CR rats exhibited age- and diet-related decreases in UDS from the AL and young CR animals, respectively. Moreover, ARO-induced old AL- and CR-derived hepatocytes exhibited significant increases in UDS compared to uninduced old AL and CR animals. No cytotoxicity was observed in the uninduced old AL- or any CR-derived hepatocytes. These data indicate that the aged and CR fed animals is less susceptible to the cytotoxic and genotoxic effects of THA; while the younger AL fed and enzyme induced old AL or CR fed animals were more susceptible. The data suggest that THA may be a genotoxic rodent carcinogen. At present, the relationship of these findings to the clinical use of THA are unclear and further study is required.

Failure to confirm increase in unscheduled DNA synthesis in sonicated mammalian cells in vitro

Two in vitro mammalian cell lines (HeLa, mouse L5178Y) were exposed/sham exposed to ultrasound (2.3 or 2.6 MHz, I-SPTA 35 W cm −2, 10 μs burst duration, 200 Hz pulse repetition frequency) for 20 min and susbequently autoradiographically scored for unscheduled DNA synthesis (UDS). A positive control (20 and 40 J m −2 germicidal ultraviolet at a flux density of 1 J m −2 s −1) yielded statistically significant increases in UDS; no such increase was observed for the ultrasound regimens. The results fail to confirm an earlier report by Liebeskind et al. of ultrasound-induced increases in UDS.

Tritiated Thymidine Incorporation into DNA in Rat Gastric Mucosal Cells

The parietal cell acid pump inhibitor, omeprazole, has undergone numerous genotoxicity studies, the conclusions of which have all been negative. A recent report by Burlinson described a method which is claimed to measure unscheduled DNA synthesis (UDS) in gastric mucosa, based on the incorporation of tritiated thymidine (3H-TdR) into DNA. In a subsequent letter it was concluded that omeprazole induces a significant increase in UDS, and it was suggested that 'for omeprazole, a genotoxic action cannot be discounted'. The Burlinson method of measuring UDS relies upon selective separation of non-dividing oxyntic mucosal surface cells from the stem cells located in the proliferative zone. These stem cells undergo normal cell division, subsequently migrate to the top and bottom of the gland, differentiating into the various specialized cells of the gland such as surface, parietal and chief cells. In the oxyntic mucosa the majority of stem cells are located at a level corresponding to one fifth of the mucosal thickness from the surface. The purpose of this study was to evaluate the Burlinson method, which depends upon the selective isolation of non-dividing surface cells by pronase digestion. The fraction used for measurement was investigated in terms of 3H-TdR uptake, the cellular distribution of 3H-TdR by autoradiography, the number of parietal cells and the sensitivity of 3H-TdR uptake for hydroxyurea (an inhibitor of normal, scheduled DNA synthesis). The results show that pronase digestion did not selectively isolate surface epithelial cells and that the digest contained both parietal cells (15 +/- 1.5%) and glandular fragments.(ABSTRACT TRUNCATED AT 250 WORDS)

The influence of dietary restriction on DNA repair in rodents: A preliminary study

A range study was undertaken to determine if dietary restriction (DR) affects DNA repair in rodents. Unscheduled DNA synthesis (UDS) was examined in two strains of rat (Brown Norway, BN and Brown Norway × Fischer 344 F 1 hybrid, BNF) at 18 months of age. O 6-Methylguanine-acceptor protein activity (MGAP) was measured across species using rat (Brown Norway × Fischer F-344 F 1 hybrid, 18 months) and mouse (B6CB F 1 hybrid, 30 months). The rodents were maintained on either an ad libitum (AL) or a restricted diet (60% of the caloric intake of AL rodents). UDS increased approximately 48–65% in freshly isolated skin cells from DR animals opposed to their AL controls after challenge with ultraviolet light (254 nm, 20 J/m 2 UV). After treatment with methylmethane sulfonate (0.5 mM MMS), a significant increase in UDS was observed ( P < 0.01, approx. 55% for BN and 52% for BNF rats). Results of measurements for MGAP activity found levels to increase 73% in DR rats and approximately 28% in DR mice when compared to their AL counterparts. In addition MGAP levels in phase shifted mice were examined at three time points during a 24-h period where significant changes were found to occur in the metabolism of DR rodents. The activity of MGAP changed in a circadian fashion with significant increases in MGAP activity in DR mice occurring during the period of highest metabolic activity.

UV-Induced unscheduled DNA synthesis in human skin: Dose response, correlation with erythema, time course and split dose exposure in vivo

Unscheduled DNA synthesis (UDS) has been shown to be saturated above a threshold dose of UV-C in human fibroblasts in vitro. We have investigated by autoradiography whether a similar saturation occurs in human skin in vivo with UV-B and whether this phenomenon correlates with the erythemal response. In addition, we determined the time course of UDS at 24 h after exposure and the effect of dual exposures separated by 24 h. The dose-response curve was established by exposure to 1/16, 1/8, 1/4, 1/2, 1, 2, 3, 4 and 6 MEDs UV-B. For the time-course study, areas exposed to 1/2 and 2 MEDs were biopsied after 1, 3, 6, 12 and 24 h. Autoradiography was performed in vitro. The dose-response curve showed a significant increase in UDS from 1/16 to 1 minimal erythema dose (MED), whereas no significant difference was observed between 1 MED and the higher UV-B doses tested. The 24 h time sequence revealed a gradual decrease in UDS activity. The 1/2 MED curve declined more rapidly and reached the zero-level between 12 h and 24 h, whereas about 50% of the initial UDS value was still retained 24 h after 2 MEDs. The dual-dose study revealed that a second hit of fractions of the MED resulted in lower levels of UDS than induced by these fractions alone in previously untreated areas. UDS increases with the erythemal dose between 1/16 and 1 MED. It reaches a plateau after 1 MED and cannot be increased by doses up to 6 MEDs, suggesting a saturation of excision repair in vivo. Time course studies support such a saturation phenomenon. The failure to increase significantly UDS by a second irradiation 24 h after the first exposure needs further clarification. Since persistence of DNA lesions may lead to an accumulation after repeated exposures, additional mechanisms other than excision repair may protect human skin by error-free removal of possibly mutagenic sites. Photo-reactivation may be important in this respect.

Modulation of ultraviolet light-, ethyl methanesulfonate-, and 7,12-dimethylbenz[a]anthracene-induced unscheduled DNA synthesis by retinol and retinoic acid in the primary rat hepatocyte.

The effects of retinol and retinoic acid on unscheduled DNA synthesis (UDS) in primary Sprague-Dawley rat hepatocytes were studied in the presence and absence of known chemical and physical mutagens. Neither retinol nor retinoic acid caused a significant increase in UDS over solvent control at concentrations ranging from 1 microM to 50 microM. Retinol and retinoic acid did not significantly affect 200 micrograms/mL ethyl methanesulfonate(EMS)- or 32 J/m2 ultraviolet light(UV)-induced UDS at concentrations ranging from 1 microM to 50 microM. In contrast, retinol and retinoic acid significantly inhibited 2.5 micrograms/mL and 5.0 micrograms/mL 7,12-dimethyl-benz[a]anthracene(DMBA)-induced UDS at concentrations of 1 microM or greater. Retinol- and retinoic acid-induced hepatocytotoxicity was studied in vitro using lactate dehydrogenase (LDH) release as an indicator of cytoxicity. Neither retinol nor retinoic acid caused significant increases in LDH release over solvent control 3 hours after treatment, whereas retinol caused a biologically significant increase in LDH release 24 hours posttreatment at concentrations of 50 microM and 100 microM. These data suggest that nontoxic concentrations of retinol and retinoic acid do not inhibit the DNA excision repair process but apparently affect the effective DNA adduct load due to the ultimate species of DMBA metabolite responsible for hepatocellular DNA damage.

Induction of hepatic cell proliferation and unscheduled DNA synthesis in mouse hepatocytes following in vivo treatment

We have modified the in vivo-in vitro hepatocyte DNA repair assay for measurement of unscheduled DNA synthesis (UDS) and hepatic cell proliferation in B6C3F1 mice. Dimethylnitrosamine and methylmethane sulfonate produced significant increases in UDS in both rats and mice. 2-Acetylaminofluorene induced a significant increase in UDS in rats, but not in mice. The mouse hepatocarcinogens, carbon tetrachloride, trichloroethylene, polybrominated biphenyls and 2,6-dichloro-p-phenylenediamine all failed to induce UDS in male and female mice, but all induced significant elevations in hepatic cell proliferation. Increased cell turnover in the liver may therefore be an important mechanism in hepatocarcinogenicity in the mouse.

Carcinogen induced unscheduled DNA synthesis in mouse hepatocytes.

Mouse primary liver cell cultures were examined for evidence of unscheduled DNA synthesis (UDS) following treatment with the carcinogens; dimethylnitrosamine (DMNA), diethylnitrosamine (DENA), 2-acetylaminofluorene (2-AAF), N-methyl-N'-nitro-N-nitrosoguanidine (MNNG), benzo(a)pyrene (BP), dimethylbenzanthracene (DMBA), 1,1,-bis(p-chlorophenyl)-2,2,2-trichloroethane (DDT), safrole, diethylstilbestrol (DES), aflatoxin B1 (AFB1), and dieldrin and the noncarcinogens; dimethylformamide (DMF), fluorene, and pyrene. Mouse hepatocyte cultures were simultaneously treated with three concentrations of each compound and 3H-thymidine. After 24 hrs, cells were fixed and processed for autoradiography. 3H-thymidine incorporation in both experimental and control cell nuclei, as evidenced by autoradiographic grains, was quantitated microscopically. DMNA, DENA, 2-AAF, MNNG, BP, AFB1 and DMBA significantly increased UDS over untreated cells at all concentrations studied. DDT, DMF, fluorene, pyrene, safrole, DES, and dieldrin were negative for UDS in all concentrations examined. DMNA, 2-AAF and MNNG were also studied for UDS induction in 2 hr old, 1 day old and 4 day old cultures. A progressive decrease in UDS with increased time after plating was found in DMNA and 2-AAF treated cultures. After 4 days DMNA and 2-AAF induced UDS only at the highest concentrations examined (10(-3) M and 10(-4) M respectively). MNNG induced UDS at all time periods and concentrations sampled. An attempt to enhance the sensitivity of the UDS assay by inducing the mixed function oxidative enzyme activity in the hepatocytes with phenobarbital administered in vivo resulted in no statistically significant increase in UDS with DMNA, 2-AAF, MNNG, DDT, and dieldrin when compared with cells from non-induced animals.

UKEMS trial compounds: Induction of unscheduled DNA synthesis in HeLa S3 cells

4CMB, 4HMB and BC were assayed for their ability to induce unscheduled DNA synthesis (UDS) in HeLa S3 cells. 4CMB elicited a positive response in the presence and absence of S9; 4HMB and BC produced no significant increase in UDS.