Unscheduled DNA Synthesis In Rat Hepatocytes Research Articles

The pathogen reduction treatment of platelets with S-59 HCl (Amotosalen) plus ultraviolet A light: Genotoxicity profile and hazard assessment

Despite restrictive donor criteria and screening procedures, infections resulting from the transfusion of bacterially contaminated platelet products continue to occur. Pathogen reduction technologies targeting nucleic acids have been developed. However, concerns about the safety of these procedures exist; the main concern being the possible mutagenic and carcinogenic effects of the pathogen-inactivated preparation in the recipient. This report reviews the genotoxicity profile of the S-59 (Amotosalen) plus long wavelength ultraviolet light (UVA) pathogen reduction technology, and assesses the mutagenic and carcinogenic hazards in recipients of treated platelets. S-59, a synthetic heterocyclic psoralen, non-covalently intercalates into the nucleic acids of pathogens and forms crosslinks when UVA photoactivated. Before clinical use, the levels of residual S-59 and free photoproducts are greatly reduced using a ‘compound adsorption device’ (CAD). In vitro, S-59 is mutagenic in Salmonella typhimurium and mouse lymphoma L5178Y TK +/− cells, and is clastogenic in CHO cells. There is reduced activity (Salmonella, CHO cells) or no activity (mouse lymphoma cells) with metabolic activation (S9 mix). When tested up to toxic dose levels, S-59 was negative in the mouse bone marrow micronucleus assay and the rat hepatocyte unscheduled DNA synthesis (UDS) test. Based on comparative studies conducted with S-59 plus UVA-treated platelets (up to 25 times without CAD), any genotoxic effects can be attributed to residual S-59. Considering (1) the known genotoxic mechanism of action for S-59, (2) the negative in vivo studies for S-59 at multiples >40,000× over clinical peak plasma levels, and (3) the fact that the positive in vitro genotoxicity effects for the end product seem due to residual S-59, any mutagenic hazard to a recipient of S-59 plus UVA-treated platelets is negligible and there is no concern about a carcinogenic potential as a consequence of a mutagenic activity. This conclusion is supported by a negative p53 +/− mouse carcinogenicity study.

Lack of DNA damage induction by okadaic acid, a marine toxin, in the CHO-Hprt and the in vitro UDS assays

Okadaic acid (OA) is a marine toxin produced by dinoflagellates and responsible for human intoxications. OA is a specific inhibitor of serine/threonine protein phosphatases PP1 and PP2A and a potent tumor promoter in mouse skin and rat glandular stomach. In a previous study, we demonstrated that OA induced aneuploidy in CHO-K1 cells using the cytokinesis-block micronucleus (CBMN) assay coupled to FISH and concluded that OA was not a direct mutagen. As some previous in vitro mutagenicity studies had given positive results with OA, we decided to perform two additional in vitro mutagenicity assays in accordance with the OECD guidelines: (i) the CHO/Hprt test, which provides end points about locus-specific gene mutation; (ii) the in vitro unscheduled DNA synthesis (UDS) assay in rat hepatocytes, which measures [ 3H]thymidine incorporation into DNA undergoing excision repair. In the CHO/Hprt assay, there was no significant increase in the number of mutants for doses ranging from 5 to 5000 nM in the presence or absence of rat liver S9 fraction. In the in vitro UDS assay, OA did not induce primary DNA damages in rat hepatocytes following 18 h exposure at concentrations between 1.32 and 100 nM. As OA could affect the DNA repair systems via the inhibition of protein phosphatases, its effects on the repair kinetic of 2AAF-induced DNA damage were also investigated with the UDS assay. The results showed that OA did not interact with the DNA-repair process involved in in vitro UDS in rat hepatocytes. We concluded that OA failed to induce direct DNA damage but acted principally by altering the chromosome number, which could contribute to its carcinogenic effect.

Genotoxic activity of extractable organic matter from urban airborne particles in Shanghai, China

The aim of this research is to investigate the impact of air pollution on the population in Shanghai. The genotoxicity of extractable organic matter (EOM) from the air particles was investigated by the means of the Salmonella plate incorporation assay, rat hepatocyte unscheduled DNA repair assay, and mice micronuclei test. The airborne particles were collected in 13 locations during the summer of 1992 and winter of 1993. The crude extracts were fractionated by acid–base partitioning into acid, base and neutral fractions. The neutral fractions were further fractionated by resin–silica gel column chromatography into three subfractions. The induction of revertants with the crude extracts was higher in winter samples than in summer samples. Both indirect-acting and direct-acting mutagenicity were observed. The mutagenicity was detected with TA98, but was not detected with TA100. The mutagenic activity was the greatest in the acid, aromatic and polar fractions from summer samples. The fractions from the winter samples did not show clear differences. There was no substantial location-related variance in the mutagenic potencies of EOM, but substantial location- or time-related variances in the mutagenic potencies of the airborne particles per cubic meter air were found. While rat hepatocyte unscheduled DNA synthesis (UDS) assay revealed genotoxicity for all the samples, there was no big variance in the genotoxicity of the fractions. The mouse micronuclei test showed results similar to the UDS assay. The difference of locality did not have statistical significance.

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.

Toxicity and health effects of vehicle emissions in Shanghai

In China, the number of vehicles is increasing rapidly with the continuous development of economy, and vehicle emission pollution in major cities is more serious than ever. In this article, we summarized the results of a series of short-term assays, animal experiments and epidemiology investigations on the genotoxicity, immunotoxicity, respiratory toxicity and health effects of vehicle emissions in Shanghai, including gasoline exhausts (gas condensate and particles), diesel exhaust particles (DEP) and scooter exhaust particles (SEP). The results showed that: (1) Both gases and particulate phases of the exhausts of different kinds of vehicles showed strong mutagenicity in Ames test (TA98 and TA100 strains), rat hepatocyte unscheduled DNA synthesis (UDS) assay, and mouse micronucleus assay, and vehicle emissions could induce the transformation of Syrian hamster embryo (SHE) cells. DEP and SEP could induce the transformation of human diploid cell strain (KMB-13) cells, immunohistochemistry assay showed that c-myc and p21 proteins were highly expressed in the transformed cells. DEP and SEP could also inhibit the gap junctional intercellular communication (GJIC) of BALB/C3T3 cells (2) Vehicle emissions could decrease the number of macrophages in the lung (bronchial alveolar lavage fluid) (BALF) of male SD rats. Vehicle emissions could also increase the proportion of polymorphonuclear leukocytes (PMN), the content of cetyneuraminic acid (NA), the activity of lactate dehydrogenase (LDH), alkali phosphate (AKP), acid phosphate (ACP) in the lung BALF of the animals. (3) In epidemiology investigation, the proportion of those who have respiratory symptoms and chronic obstructive pulmonary diseases (COPD) in the workers who were exposed to DEP ( n=806) were much higher than those of the controls ( n=413). The OR (odd ratio) values of angina, nasal obstruction, phlegm, short of breath and COPD were 2.27, 3.08, 3.00, 3.19 and 2.32, respectively, and the proportion of those who have indisposed sensation of nose or laryngopharynx, cough, phlegm and pharyngitis in the workers who were occupationally exposed to gasoline exhausts ( n=157) were also higher than those of controls ( n=121), the OR values were 2.43, 3.76, 2.58, and 3.70, respectively, and in the 40 gasoline exhausts exposed workers, the frequencies of 6-TG (thioguanine), sister chromatid exchanges (SCEs) and micronuclei in peripheral blood were markedly higher ( P<0.05) than those of controls. The SI (T lymphocytes transformation) activity, total E rosette, E active rosette, content of immunoglobulin A (IgA) and fibrin (FN) of the exposed group were significantly ( P<0.05) decreased compared with those of the control. All the results showed that vehicle emissions could not only induce adverse effects on respiratory and immune system of occupationally exposed people, but also have potential carcinogenicity to human beings.

Bioassay of quinoline, 5-fluoroquinoline, carbazole, 9-methylcarbazole and 9-ethylcarbazole in newborn mice

Quinoline and carbazole are among the more prevalent aza-arenes present as components of environmental pollutants. Both of these aza-arenes are hepatocarcinogenic to mice when administered in the diet. The hepatocarcinogenic potential of quinoline is consistent with its mutagenic activity in Salmonella typhimurium TA100 and potential to induce unscheduled DNA synthesis (UDS) in rat hepatocytes. Structure-activity studies with fluorinated quinolines indicate that the presence of a fluorine atom at the 5-position of quinoline may inhibit detoxification and result in enhanced genotoxic potency. Quinoline and 5-fluoroquinoline were assayed in newborn CD-1 mice at a total dose of 1.75 μmol to establish their relative tumorigenic activity. Liver tumours developed in 60 and 90% of the male newborn mice treated with quinoline and 5-fluoroquinoline, respectively. The majority of liver tumours observed among the quinoline-treated mice were classified as adenomas. In contrast, liver carcinomas developed in most of the male mice treated with 5-fluoroquinoline. Unlike the well established genotoxic potential of quinoline, there is limited evidence for carbazole having either genotoxic or carcinogenic activity. Whereas carbazole is not mutagenic towards several strains of S. typhimurium, both 9-methylcarbazole and 9-ethylcarbazole are active as mutagens in S. typhimurium TA100. Carbazole, 9-methylcarbazole 9-ethylcarbazole were assayed in primary rat hepatocytes to assess their relative potential to induce UDS in rat hepatocytes; only 9-ethylcarbazole did so. These carbazole derivatives were also assayed in newborn CD-1 mice at a total dose of 1.75 μmol. Neither carbazole nor either of these 9-alkylcarbazoles produced a significant tumorigenic response in this bioassay system.

DNA modification and repair by 2-nitropropane is extensive in hepotocytes of rats compared to those of humans and mice

Hepatocytes from rats, mice or humans were exposed to either 2-nitropropane or propane 2-nitronate and the extent of unscheduled DNA synthesis (UDS) was measured. At a concentration of 2-nitropropane (0.1 mM) that produced a marked induction of UDS in rat hepatocytes, a negative or minimal response from mice and humans. A 10-fold higher concentration of 2-nitropropane was required in mouse hepatocytes for an equivalent UDS response to that seen in rat-liver cells. All three species showed UDS after exposure of hepatocytes to propane 2-nitronate at 0.1 mM although the effect in human cells was variable. In rat hepatocytes the induction of UDS by 2-nitropropane was not inhibited by the antioxiodant dimethyl sulphoxide (1%). In these cells, 2-nitropropane produced an electrochemically active modified deoxynucleoside, similar to that reported to be formed in livers of rats which had received 2-nitropropane in vivo. A smaller but significant production of this altered deoxynucleoside was found in mouse hepatocytes but not in human hepatocytes treated identically. The level of 8-oxo7,8-dihydrodeoxyguanosine was not increased in hepatocytes from any of the three species exposed to 2-NP under the same conditions. It is suggested that the UDS caused by 2-nitropropane may be in response to damage other than that produced by oxygen radicals.

Evaluation of the potential of riddelliine to induce unscheduled DNA synthesis, S-phase synthesis, or micronuclei following in vivo treatment with multiple doses.

Riddelliine (RID) is a pyrrolizidine alkaloid found in plants of the genera Crotalaria, Amsinckia, and Senecio in the United States. RID has been extensively studied in a wide variety of in vitro short-term genotoxicity tests and has yielded positive responses in most test systems; however, there are fewer data available on the effects of RID in in vivo assays and no data under repeat-dose regimens. We have evaluated the ability of RID to induce unscheduled DNA synthesis (UDS) in hepatocytes, S-phase synthesis (SPS) in hepatocytes, and micronuclei in bone marrow from animals dosed for 5 or 30 days in conjunction with prechronic toxicity testing conducted for the National Toxicology Program. Results of this study indicate that RID did not induce an increase in micronucleated polychromatic erythrocytes (PCE) in bone marrow of mice after 5 days of dosing or in PCE from rats or mice after 30 days of dosing. RID did not induce an increase in UDS in rat hepatocytes after 5 or 30 days of dosing, but it did induce an equivocal UDS response in male mice after both time points and a positive response in female mice after 30 days of dosing. RID induced significant elevations in SPS in rat hepatocytes after both 5 and 30 days of dosing, even at low doses. An increase in SPS was observed in male and female mouse hepatocytes, but only after 30 days of dosing. Rats and mice of both sexes showed a depression in SPS at higher doses. This effect may be a result of toxicity, which compromises the ability of the liver to regenerate. These results demonstrate that repeated administration of test chemicals may alter the genotoxic response to chemicals.

Unscheduled DNA synthesis in human hair follicles after in vitro exposure to 11 chemicals: comparison with unscheduled DNA synthesis in rat hepatocytes

A new method is described to investigate unscheduled DNA synthesis (UDS) in human tissue after exposure in vitro: the human hair follicle. A histological technique was applied to assess cytotoxicity and UDS in the same hair follicle cells. UDS induction was examined for 11 chemicals and the results were compared with literature findings for UDS in rat hepatocytes. Most chemicals inducing UDS in rat hepatocytes raised DNA repair at comparable concentrations in the hair follicle. However, 1 of 9 chemicals that gave a positive response in the rat hepatocyte UDS test, 2-acetylaminofluorene, failed to induce DNA repair in the hair follicle. Metabolizing potential of hair follicle cells was shown in experiments with indirectly acting compounds, i.e., benzo[ a]pyrene, 7,12-dimethylbenz[ a]anthracene and dimethylnitrosamine. The results support the conclusion that the test in its present state is valuable as a screening assay for the detection of unscheduled DNA synthesis. Moreover, the use of human tissues may result in a better extrapolation to man.

Genotoxic properties of ( E)-5-(2-bromovinyl)-2′-deoxyuridine (BVDU)

( E)-5-(2-bromovinyl)-2′-deoxyuridine (BVDU) is a 5-substituted 2′-deoxyuridine antiviral compound that inhibits thymidylate synthetase. The selectivity of BVDU for virus-infected cells has been attributed to phosphorylation of BVDU by a virus-induced thymidine kinase. Since the closely related compounds 5-bromo-2′-deoxyuridine and 5-iodo-2′-deoxyuridine are in vitro and in vivo mutagens, BVDU was tested for genotoxic activity in bacterial and mammalian cell mutation assays as well as in assays measuring DNA damage/repair and clastogenic activity. Mutation assays with BVDU at concentrations ranging from 10 to 5000 μg/plate using Salmonella typhimurium strains TA1535, TA1537, TA1538, TA98, and TA100 were negative, both with and without S9 activation. BVDU was also negative in the in vitro rat hepatocyte unscheduled DNA synthesis assay at concentrations of 750 and 1000 μg/ml. In contrast, BVDU was positive in the L5178Y TK ± mouse lymphoma mutation assay without S9 activation at five concentrations ranging from 500 to 2000 μg/ml. A Chinese hamster ovary cell (CHO)/hypoxanthine guanine phosphoribosyl transferase gene mutation assay conducted without S9 over similar concentrations was negative. However, micronucleus induction by BVDU was detected without S9 activation at concentrations between 500 and 1750 μg/ml using both CHO and L5178Y cells. These results indicate that BVDU is a potential human clastogen.

Strain differences in in vitro rat hepatocyte unscheduled DNA synthesis (UDS): Effect of UV is independent of strain while increased sensitivity is apparent using Fischer-344 instead of Sprague-Dawley rats

The unscheduled DNA synthesis (UDS) assay measures DNA repair following in vitro treatment of rat primary hepatocytes. This report compares the UDS response of primary hepatocytes from 2 widely used rat strains, the Fischer-344 (F344) and Sprague-Dawley (SD) strains. Ultraviolet (UV) light and 5 known genotoxic chemicals were evaluated in each strain in parallel experiments. The chemicals tested were 2-acetylaminofluorene (2-AAF), 4-aminobiphenyl (4-AB), benzidine, dimethylnitrosamine (DMN) and N-propyl- N′-nitro- N-nitrosoguanidine (PNNG). Four of these compounds (2-AAF, 4-AB, benzidine and DMN) require metabolic activation. Benzidine and PNNG were both negative using SD rat hepatocytes, but were weakly positive using F344 rat hepatocytes. In the first of 2 experiments, 4-AB was inconclusive in SD hepatocytes, but strongly positive in F344 cells. In the second experiment, 4-AB was positive in hepatocytes from both strains. 2-AAF was more strongly positive in F344 cells than in SD cells. DMN and UV light induced positive dose responses with little or no differences between strains. It is concluded that hepatocytes from F344 rats may be more sensitive, qualitatively and quantitatively, than hepatocytes from SD rats as indicators of UDS. This difference is not due to intrinsic differences in DNA repair mechanisms but is probably due to differences in drug-metabolizing enzymes between these strain. Thus, for routine screening, F344 rats are preferable for measurement of the in vitro UDS-inducing potential of compounds.

Genotoxicity of fluoroquinolines and methyiquinolines

Quinoline in the presence of microsomal activation exhibits mutagenic activity in Salmonella typhimurium TA100 and induces unscheduled DNA synthesis (UDS) in rat hepatocytes. Structure-activity studies were performed to determine those positions on quinoline critically associated with its genotoxicity. In assays performed to determine the ability of 2-, 4-, 6- and 8-methylquinoline to induce UDS, only 4- and 8-methylquinoline produced a positive response. This represents an improved correlation for these methylquinolines with tumorigenic activity as compared to that previously observed with mutagenic activity in S. typhimurium TA100. The complete isomeric series of fluoroquinolines were evaluated as mutagens in S. typhimurium TA100 and for their potential to induce UDS in rat hepatocytes. The only isomers that did not exhibit significant mutagenic activity were 2- and 3-fluoroquinoline. Among these isomeric fluoroquinolines 5-, 6-, 7- and 8-fluoroquinoline are capable of inducing UDS. No significant effect on UDS was observed for 2-, 3- or 4-fluoroquinoline. These data indicate that positions 1-3 in quinoline are critical sites associated with its genotoxicity.

Chemical characterization and mutagenic properties of polycyclic aromatic compounds in sediment from tributaries of the great lakes

AbstractSediments from four inshore industrial sites and a reference site in the Great Lakes were extracted with solvents and characterized chemically for polycyclic aromatic compounds (PACs). An aqueous phase and a crude organic extract were obtained. The crude organic extract was further resolved into fractions A‐2 (polycyclic aromatic hydrocarbons) and A‐3 (nitrogen‐containing polycyclic aromatic compounds), which were analyzed for PACs by gas chromatography and gas chromatography‐mass spectrometry. The extracts and fractions were tested for mutagenicity in three assays: Ames, rat hepatocyte unscheduled DNA synthesis, and Chinese hamster ovary hypoxanthine‐guanine phosphoribosyl transferase (CHO/HGPRT). Sediments from the industrial sites contained 27 to 363 μg/g total PACs; the reference site, less than 1 μg/g. Qualitative differences in the residue profiles among the sites were attributable to the probable sources of the PACs (petroleum versus combustion). Only one industrial site yielded measurable (0.1 μg/g or more) concentrations of individual nitrogen‐containing PACs. In the Ames assay, only the highest doses of the A‐2 fractions from two sites approached positive results. Conversely, the crude organic extract and A‐2 and A‐3 fractions from all sites induced unscheduled DNA synthesis. Crude organic extracts and the A‐2 and A‐3 fractions from all industrial sites gave well‐defined dose‐response relations in the CHO/HGPRT assay. We established the presence of chemical mutagens in sediment that could be correlated with neoplasms in fish from many of the sites; however, the mutagenicity of the sediment extracts was not completely related to the degree of contamination by PACs. We also discuss the utility of mutagenicity assays in the evaluation of complex chemical mixtures and recommend the use of a CHO/HGPRT‐type assay in which cells are not required to proliferate in the presence of potential interfering chemicals.

The genotoxic activity of glycerol in an in vitro test battery

Glycerol, a widely distributed constituent of food and an additive used in cigarette manufacture, has been tested for genotoxic potential in a battery of short-term genotoxicity assays. Glycerol was evaluated in the Ames Salmonella typhimurium mutagenesis assay (strains TA98, TA100, TA1535, TA1537 and TA1538), in the rat hepatocyte unscheduled DNA synthesis assay, in the Chinese hamster ovary (CHO) chromosome aberration assay, the CHO sister chromatid exchange assay and the CHO mammalian mutagenesis assay. All assays (except the rat hepatocyte unscheduled DNA synthesis assay) were conducted both with and without the addition of Aroclor-induced rat liver S-9. The results of all tests were negative, showing that neither glycerol nor its metabolites have genotoxic activity in the battery of tests used.