Unscheduled DNA Synthesis Activity Research Articles

AniFOUND: analysing the associated proteome and genomic landscape of the repaired nascent non-replicative chromatin.

Specific capture of chromatin fractions with distinct and well-defined features has emerged as both challenging and a key strategy towards a comprehensive understanding of genome biology. In this context, we developed aniFOUND (accelerated native isolation of factors on unscheduled nascent DNA), an antibody-free method, which can label, capture, map and characterise nascent chromatin fragments that are synthesized in response to specific cues outside S-phase. We used the ‘unscheduled’ DNA synthesis (UDS) that takes place during the repair of UV-induced DNA lesions and coupled the captured chromatin to high-throughput analytical technologies. By mass-spectrometry we identified several factors with no previously known role in UVC-DNA damage response (DDR) as well as known DDR proteins. We experimentally validated the repair-dependent recruitment of the chromatin remodeller RSF1 and the cohesin-loader NIPBL at sites of UVC-induced photolesions. Developing aniFOUND-seq, a protocol for mapping UDS activity with high resolution, allowed us to monitor the landscape of UVC repair-synthesis events genome wide. We further resolved repair efficacy of the rather unexplored repeated genome, in particular rDNA and telomeres. In summary, aniFOUND delineates the proteome composition and genomic landscape of chromatin loci with specific features by integrating state-of-the-art ‘omics’ technologies to promote a comprehensive view of their function.

Nucleotide Excision Repair Is Not Induced in Human Embryonic Lung Fibroblasts Treated with Environmental Pollutants

The cellular response to genotoxic treatment depends on the cell line used. Although tumor cell lines are widely used for genotoxicity tests, the interpretation of the results may be potentially hampered by changes in cellular processes caused by malignant transformation. In our study we used normal human embryonic lung fibroblasts (HEL12469 cells) and tested their response to treatment with benzo[a]pyrene (B[a]P) and extractable organic matter (EOM) from ambient air particles <2.5 µm (PM2.5) collected in two Czech cities differing in levels and sources of air pollution. We analyzed multiple endpoints associated with exposure to polycyclic aromatic hydrocarbons (PAHs) including the levels of bulky DNA adducts and the nucleotide excision repair (NER) response [expression of XPE, XPC and XPA genes on the level of mRNA and proteins, unscheduled DNA synthesis (UDS)]. EOMs were collected in the winter and summer of 2011 in two Czech cities with different levels and sources of air pollution. The effects of the studied compounds were analyzed in the presence (+S9) and absence (–S9) of the rat liver microsomal S9 fraction. The levels of bulky DNA adducts were highest after treatment with B[a]P, followed by winter EOMs; their induction by summer EOMs was weak. The induction of both mRNA and protein expression was observed, with the most pronounced effects after treatment with B[a]P (–S9); the response induced by EOMs from both cities and seasons was substantially weaker. The expression of DNA repair genes was not accompanied by the induction of UDS activity. In summary, our results indicate that the tested compounds induced low levels of DNA damage and affected the expression of NER genes; however, nucleotide excision repair was not induced.

Unscheduled DNA synthesis in human lymphocytes treated with cigarette smoke condensate and its semivolatile fraction.

A cigarette smoke condensate (CSC) and the semivolatile fraction (SVF) from the CSC were tested for their ability to induce unscheduled DNA synthesis (UDS) in human lymphocytes. Exposed cells were incubated with tritiated thymidine and the UDS was determined by liquid scintillation counting of precipitated DNA. The tests were performed with and without metabolic activation by liver fraction (S9) from rats induced with Aroclor 1254. No UDS activity was detected, neither for the CSC nor for the SVF. Two control substances, benzo(a)pyrene and methyl methanesulfonate, increased the UDS activity in tests with and without metabolic activation, respectively. The scintillometric method used for detection of UDS was not sufficiently sensitive to be used as a tool for genetic risk assessment of CSC fractions.

Kinetics of DNA adduct formation and removal in mouse hepatocytes following in vivo exposure to 5,9-dimethyldibenzo[c,g]carbazole.

5,9-Dimethyldibenzo[c,g]carbazole (DMDBC), a potent mouse hepatocarcinogen, has been shown to induce a non-linear increase in mutant frequency in the liver of the transgenic MutaMouse. To gain insight into the mechanisms underlying the mutagenicity of DMDBC in vivo, DNA damage formation and removal were monitored in mouse hepatocytes over 4-144 h after a single skin application of 10 or 90 mg/kg DMDBC. DNA adducts were measured by (32)P-post-labeling. DNA repair was assessed by: (i) the unscheduled DNA synthesis (UDS) assay, which measures [(3)H]thymidine incorporation into hepatocyte DNA undergoing excision repair; (ii) the Comet assay, which detects DNA strand breaks transiently produced between the incision and rejoining steps of the excision repair process. A plateau of approximately 400 DNA adducts/10(8) nucleotides was reached 24 h after treatment with 10 mg/kg and remained unchanged until 144 h. UDS activity was significantly induced at 15 and 24 h, while no DNA strand breaks were observed at any sampling time. These results suggest that DNA repair mechanisms were efficiently induced and the formation of a high degree of DNA damage was avoided at this dose level. Following exposure to 90 mg/kg DMDBC, the number of DNA adducts increased sharply to a maximum at 24 h ( approximately 8000/10(8) nucleotides) and then declined to approximately 500/10(8) nucleotides at 144 h. UDS activity was markedly induced from 15 to 72 h. Low levels of DNA strand breaks were observed at 24 and 48 h. The formation of large numbers of DNA adducts and the emergence of DNA strand breaks despite a strong initial induction of UDS activity suggested that DNA repair mechanisms were saturated at this dose level. This phenomenon could partly account for the non-linear induction of gene mutations previously reported in the liver of the transgenic MutaMouse.

Lack of UDS activity in the livers of rats exposed to allylisothiocyanate.

Allylisothiocyanate (AITC) has been evaluated for its ability to initiate unscheduled DNA synthesis (UDS) in the livers of male rats in vivo. Specific Pathogen Free outbred albino Hsd/Ola Sprague-Dawley rats were exposed by oral gavage to 37.5 or 125 mg/kg AITC in corn oil and hepatocytes assessed for UDS by autoradiography 2 and 14 h later. AITC did not induce UDS at either dose level at either time point. These data are consistent with all other evidence indicating that AITC does not act as a genotoxin in vivo, despite positive response in some in vitro screening assays. The reported occurrence of benign bladder papillomas in male rats but not female rats or mice of either sex is consistent with non-genotoxic action and may be attributed to chronic irritation of the bladder epithelium by AITC and its cysteine conjugate metabolite excreted by male rats in unusually concentrated form. It is concluded that the weight of evidence is insufficient to regard AITC as a genotoxin capable of human carcinogenicity.

Effect of eugenol on the genotoxicity of established mutagens in the liver

The influence of in vivo treatment with eugenol on established mutagens was studied to determine whether eugenol has antigenotoxic potential. The effects of eugenol in rats was investigated in the unscheduled DNA synthesis (UDS) assay with established mutagens and the Salmonella typhimurium mutagenicity assay. In addition, the effect of in vitro treatment with eugenol on benzo[ a]pyrene (B[ a]P)-induced genotoxicity in human hepatoma cell line Hep G2 was investigated in the single-cell gel electrophoresis assay. The mutagenicity of B[ a]P in the S. typhimurium mutagenicity assay was lower in liver S-9 fractions prepared from rats treated with eugenol orally (1000 mg/kg body weight) than in liver S-9 fractions from control rats. Incubation of liver S-9 fractions from eugenol-treated rats with dimethylbenzemthracene (DMBA) had no antimutagenic effect. Eugenol did not modify UDS activity in hepatocytes isolated from rats pretreated with eugenol orally after exposure of these cells in vitro to DMBA and aflatoxin B 1. Four different treatment schemes of combinations of B[ a]P and eugenol were examined in Hep G2 cells: pre-treatment with eugenol; simultaneous treatment with eugenol and B[ a]P; a combination of these (pretreatment/simultaneous treatment); and post-treatment with eugenol. An increase in the genotoxicity of B[ a]P was found in Hep G2 cells. No effect of eugenol on the genotoxicity of B[ a]P was found with the pre- and post-treatments. It is concluded that the effect of eugenol on genotoxicity induced by established mutagens is not univocal: in vivo treatment of rats with eugenol resulted in a reduction of the mutagenicity of B[ a]P in the S. typhimurium mutagenicity assay, while in the UDS assay no effect of eugenol was found. In vitro treatment of cultured cells with eugenol resulted in an increase in genotoxicity of B[ a]P. These findings indicate that there is only limited support for the antigenotoxic potential of eugenol in vivo.

SHORT COMMUNICATION: Time-Related introduction of DNA repair synthesis in rat hepatocytes following in vivo treatment with cyproterone acetate

Cyproterone acetate (CPA), a synthetic steroid hormone widely used as a human pharmaceutical, has recently been shown to induce unscheduled DNA synthesis (UDS) in vitro in primary cultures of rat and human hepatocytes. In the present study CPA was evaluated for its ability to initiate UDS in the liver of female rats in vivo by means of the in vivo/in vitro hepatocyte DNA repair test. Using the standard sampling time of 16 h after single oral dosing, a dose-related UDS response at >50 mg CPA/kg body wt was observed. In order to examine the time course of CPA-induced UDS, different sampling times (4, 16, 48, 72, 96 and 144 h) after a single oral administration of 100 mg CPA/kg body wt were used. Whereas no UDS was induced in liver cells isolated 4 h after treatment, continuous DNA repair activity was observed after 16 h, with a maximum effect of approximately 10 net nuclear grains on day 4 after dosing. After a post-application period of 6 days the net grain counts returned to near control level. In contrast to the data with CPA, DNA damage induced by the positive control compound 2-acetylaminofluorene was removed much faster, within 2 days after administration. The time course of UDS activity during the first 4 days after administration of CPA parallels the previously reported time course of DNA adduct formation. However, a subsequent decrease and final cessation of UDS on day 6 takes place, although significant levels of CPA-DNA adducts have been reported to occur at that time point in rat liver cells. Whereas the time-related onset of UDS is suggested to reflect the pharmacokinetics of the activation of CPA to DNA-reactive intermediates, it remains unclear why the excision repair of DNA adducts did not continue. In addition to DNA repair synthesis, CPA was also shown to clearly increase the incidence of S phase cells under the test conditions applied, confirming the known mitogenic potential of CPA in rat liver.

Recommendations for the performance of UDS tests in vitro and in vivo

The Working Group (WG) dealt with the harmonisation of routine methodologies of tests for unscheduled DNA synthesis (UDS) both in vitro and in vivo. In contrast to the existing guidelines from OECD, EPA and EC on in vitro UDS tests (there is no Japanese UDS guideline), the Working Group recommends that in general in vitro UDS tests should be performed with primary hepatocytes. For routine applications any other cell types would need special justification. Hepatocytes from male rats are preferable, unless there are contra-indications on the basis of e.g. toxicokinetic data. According to the OECD, EPA and EC guidelines, UDS may be analysed by means of autoradiography (AR) or liquid scintillation counting (LSC). The WG recommends use of AR. LSC is less suitable due to the problem of differentiation between UDS activity and replicative DNA synthesis, and the disadvantage that cells cannot be analysed individually. Since a specific cell type was recommended by the WG, methodological aspects could be described in more detail than in the present guidelines. For in vitro tests, it was agreed that the initial viability of freshly isolated hepatocytes should be at least 70%. With regard to the need for confirmatory experiments in the event of a clear-cut negative results, the majority view was that confirmation by a second (normally not identical) experiment is still needed; this is line with the present OECD and EC guidelines. Evaluation of results from UDS tests should be based primarily on net nuclear grain (NNG) values, although it is recognised that nuclear and cytoplasmic gains result from different biological processes. Since grain counts are influenced by a number of methodological parameters, no global threshold NNG value can be recommended for discrimination of postive and negative UDS results. For in vitro assays, the criteria for positive findings go beyond those of the present guidelines and two alternative approaches are given which are based on (1) dose-dependent increases in NNG values and (2) reproducibility, dose-effect relationship and cytotoxicity. At present there is no official guideline on the performance of the vivo UDS tests. Some fundamental recommendations given for in kvitro methodology also apply to the in vivo assay. For routine testing with the in vivo UDS tests, again the general use of hepatocytes from male rats is recommended. However, concerning the requirement to use one or two sexes, consistency with other in vivo genotoxicity assays (e.g. the micronucleus assay) would be preferable. As for the in vitro methodolody, AR is preferred rather than LSC. For in vivo UDS tests, a minimum viability of 50% is considered to be sufficient. Sampling of cells 12–16 h after treatment and, if this is negative, 2–4 h recommended. At least three animals per treatment group should be used. Evaluation of results should again be done on the basis of NNG values. The fundamental criterion for a positive result is given by an increase of NNG values for at least one experimetnal group. This NNG increase should be evaluated by consideration of (1) lab-specific historical controls or adequate statistics, or (2) interanimal variation, dose-effect relationship and cytotoxicity.

Two types of proliferating cell nuclear antigen (PCNA) complex formation in quiescent normal and xeroderma pigmentosum group A fibroblasts following ultraviolet light (uv) irradiation

We examined the relationship between the formation of proliferating cell nuclear antigen (PCNA) complex with DNA and nucleotide excision repair in human fibroblasts following ultraviolet light (uv) irradiation. PCNA complex formation was detected by the immunofluorescence method after methanol fixation and nucleotide excision repair activity was detected as the unscheduled DNA synthesis (UDS) by autoradiography labeled with [ 3H]thymidine. Quiescent normal cells showed a strong punctuated pattern of PCNA staining 5 min to 3 h and UDS 3 h after 10 J/m 2 of uv irradiation, but they no longer showed PCNA staining and UDS 24 h after irradiation. In contrast, xeroderma pigmentosum group A (XP-A) cells, which lack UDS activity, did not show PCNA staining up to 30 min after irradiation; however, unexpectedly, they were stained 3 h and even 24 h after irradiation with their staining pattern being different from that in normal cells. Namely, the fluorescence spots in XP-A cells were larger in size and much smaller in number than those in normal cells. When XP-A cells were fused with normal cells with polyethylene glycol treatment, nuclei of XP-A cells showed a PCNA staining pattern similar to that of normal cells at 30 min, which was no longer detected 24 h after irradiation. These results suggest that there exist two types of PCNA complex formation, nucleotide excision repair-related and -unrelated, in human fibroblasts following uv irradiation.

Enhanced unscheduled DNA synthesis by secondary cultures of lung cells established from calorically restricted aged rats

Unscheduled DNA synthesis (UDS) induced by two exposure levels of ultraviolet light (UV) or two concentrations of methyl methane sulfonate (MMS) was evaluated in secondary cultures of lung fibroblasts established from weanling, 11-month-old and 31-month-old female Fischer 344 rats fed and libitum (AL) or calorically restricted (CR) diets. [ 3H]Thymidine incorporation as a function of UDS was highest for weanling-derived cells treated with either UV or MMS, declining consistently with increased age between cells from weanling, 11-month-old and 31-month-old animals. [ 3H]Thymidine incorporation as a function of UDS in cells from 11-month-old AL vs. CR rats differed only at the highest UV exposure level. In contrast, cells drived from 31-month-old CR rats exhibited UDS levels which were at leastt wice as high at each UV treatment level as UDS levels of cells drived from the same age AL rats. Cells from both old AL and old CR rats were shown to initiate DNA excision repair at about the same rate. Cells from CR rats, however, repaired DNA damage at an accelerated rate and completed excision repair while repair in cells from AL animals was slower and apparently did not proceed to completion. Data from this study indicate that cells from young and old AL and CR animals initiate excision repair, but demonstrate an age-related loss of UV- or MMS-stimulated [ 3H]thymidine incorporation in cells derived from AL animals. Cells derived from CR animals did not exhibit that age-related loss of UDS activity; rather, they showed an enhanced UDS response to DNA damage and appeared to complete ligation as the final step in excision repair. The data suggest that caloric restriction of a cell donor animal not only delays the age-associated decrease in in vitro DNA excision repair capacity in cells from that animal, but may actually enhance repair capacity.

Quinoline: Unscheduled DNA synthesis and mitogenesis data from the rat liver in vivo

Quinoline is a specific and potent carcinogen to the rat and mouse liver. Studies are described here in which it was evaluated for its ability to initiate unscheduled DNA synthesis (UDS) in the rat liver in vivo. Although some individual animals showed indications of a marginal response the absence of clear group positive responses and the lack of an obvious dose relationship precluded the classification of quinoline as positive. The analogous NTP non-carcinogen 8-hydroxyquinoline was shown also to be devoid of UDS activity. Quinoline did, however, induce a potent mitogenic response in the rat liver between 24 and 48 hr after oral dosing of 200-500 mg/kg. Under similar conditions of test, 8-hydroxyquinoline was essentially inactive. These data represent a further instance in which mitogenicity in the liver appears to correlate better with carcinogenicity than does genotoxicity; but it may not be that simple, as discussed in the text. A single dose of quinoline was shown to act as a replacement for surgical partial hepatectomy in the liver micronucleus assay described by Tates, consistent with its potent mitogenicity.

Multiple abnormalities in the ultraviolet light response of cultured fibroblasts derived from patients with the basal cell nevus syndrome.

Basal Cell Nevus Syndrome (BCNS) is a rare autosomal-dominant inherited disorder associated with a marked hypersusceptibility to spontaneous and radiation-induced skin cancer. We examined the changes in cell survival, unscheduled DNA synthesis (UDS) and the frequency of sister chromatid exchanges (SCE) induced by ultraviolet light (UVL) in confluent normal and BCNS fibroblasts. BCNS cells appeared slightly hypersensitive to the cytotoxic effects of UVL. The rate of UDS induced by UVL exposure in normal cell strains increased linearly following doses up to 30 J/m2, whereas in BCNS cells UDS became saturated at doses of 10 J/m2 showing no further increase with doses up to 30 J/m2. UDS activity persisted for longer periods after UVL exposure in BCNS as compared with normal cells. The dose-response relationship for UVL-induced SCE was similar in normal and BCNS fibroblasts. However, the frequencies of UVL-induced SCE declined to near background levels in normal cells following 12-24 hr of confluent holding prior to subculture whereas they remained elevated in BCNS cells with holding times up to 24 hr after UVL exposure. Overall, these results suggest that BCNS fibroblasts may have a diminished capacity for the repair of some type of DNA damage as compared with normal fibroblasts.

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.

Influence of age, sex and strain on the in vitro induction of unscheduled DNA synthesis in rat hepatocyte primary cultures.

The activity of chemical-induced unscheduled DNA synthesis was evaluated in hepatocyte primary cultures from Fischer 344 and Sprague-Dawley rats over a period of two years. In this two-year study hepatocytes from both sexes and strains were prepared from animals 2, 8, 14, 20 and 25 months of age and UDS was measured by autoradiography following treatment with N-methyl-N'-nitro-N-nitrosoguanidine and 2-acetylaminofluorine. A dose-related positive response occurred for both compounds throughout the study in hepatocytes from male and female Fischer rats and male Sprague-Dawley rats. The magnitude of the response was greatest in hepatocytes from male Fischer rats and a markedly lower response in unscheduled DNA synthesis occurred in all cultures prepared from animals of both strains and sexes at 20 and 25 months of age. Hepatocytes from female Sprague-Dawley rats showed a low level of unscheduled DNA synthesis with N-methyl-N'-nitro-N-nitrosoguanidine throughout the study. The most striking finding was the absence of a UDS response to 2-acetylaminofluorene by hepatocytes from Sprague-Dawley females at the 8, 14, 20 or 25 month periods. The results indicate an age-related decrease in chemical-induced unscheduled DNA synthesis activity among rats.

Assessment of unscheduled and replicative DNA synthesis in rat kidney cells exposed in vitro or in vivo to unleaded gasoline

Unleaded gasoline (UG) induces renal toxicity and neoplasia in male but not female rats after chronic inhalation exposure. Before a meaningful determination of the potential human health risk of UG can be made, it is imperative that the mechanism responsible for its carcinogenic action be understood. The purpose of the present investigation was to determine whether the induction of kidney tumors by UG is related to genotoxic or to cell-proliferative effects. Unscheduled DNA synthesis (UDS), as an indicator of genotoxicity, was measured autoradiographically as the incorporation of [ 3H]thymidine in isolated rat kidney cells following in vivo or in vitro exposure to UG. As an indicator of proliferative activity, cells in S-phase were quantitated in isolated cell preparations obtained from exposed rats. UG was administered to rats by inhalation (2000 ppm) or by gavage (up to 5000 mg/kg). The ability of the in vivo/in vitro kidney cell UDS assay to detect genotoxicants was verified using a variety of compounds. No UDS activity was elicited by UG under any of the conditions employed, including inhalation exposure to a concentration that produced kidney tumors in the 2-year bioassay. A five- to eightfold increase in the percentage of cells in S-phase was observed in male rats exposed to UG for 18 days either by inhalation or by gavage. Cell turnover was not markedly enhanced in identically treated female rats. These data indicate that UG does not evoke UDS in the rat kidney even after exposures that, in all probability, resulted in greater tissue concentrations of UG components than was realized in the long-term inhalation bioassay. The sex-specific induction of replicative DNA synthesis in the kidney paralled the carcinogenic activity of UG, suggesting that induced cell turnover may be an important factor in the carcinogenic action of this motor fuel.

Mutations at the mei-41, mus(1)101, mus(1)103, mus(2)205 and mus(3)310 loci of Drosophila exhibit differential UDS responses with different DNA-damaging agents

5 mutagen-sensitive mutants of Drosophila melanogaster, reported to perform normal or only slightly reduced excision repair of UV damage, were examined by an unscheduled DNA synthesis (UDS) assay. This assay measures the ability of cultured primary cells, derived from each mutant, to perform the resynthesis step in the excision repair pathway, following damage to cellular DNA by direct-acting alkylating agents, UV or X-irradiation. 2 mutants, classified as completely or partially proficient for both excision and postreplication repair of UV damage, mus(1)103 and mus(2)205, were found to give positive UDS responses only for UV damage. These mutants exhibit no measurable UDS activity following DNA damage by several different alkylating agents and X-rays. 3 mutants, classified as having no defect in excision repair, but measurable defects in postreplication repair of UV damage, mei-41, mus(1)101, and mus(3)310 exhibit 3 different response patterns when tested with the battery of agents in the UDS assay. The mutant mei-41 exhibits a highly positive UDS response following damage by all agents, consistent with its prior classification as excision-repair-proficient, but postreplication-repair-deficient for UV damage. The mutant mus(1)101, however, exhibits a strong positive UDS response following only UV damage and appears to be blocked in the excision repair of damage produced by both alkylating agents and X-irradiation. Finally, mus(3)310 exhibits no UDS response to alkylation, X-ray or UV damage. This is not consistent with its previous classification. Results obtained with the qualitative in vitro UDS assay are entirely consistent with the results from two separate in vivo measures of excision repair deficiency following DNA damage, larval hypersensitivity to killing and hypermutability in the sex-linked recessive lethal test.

Assessment of unscheduled and replicative DNA synthesis in hepatocytes treated in vivo and in vitro with unleaded gasoline or 2,2,4-trimethylpentane

In a recent chronic inhalation exposure study, unleaded gasoline (UG) produced kidney tumors in male rats and liver tumors in female mice, but did not increase the incidence of liver tumors in male mice or rats of either sex. To examine the possible basis for this pattern of hepatocarcinogenesis, unscheduled DNA synthesis (UDS) as an indicator of genotoxic activity and replicative DNA synthesis (RDS) as an indicator of cell proliferation were measured in rat and mouse hepatocytes following in vivo and in vitro exposures to UG and 2,2,4-trimethylpentane (TMP), a nephrotoxic component of UG. Primary hepatocyte cultures, prepared from cells isolated from Fischer-344 rats, B6C3F1 mice, or human surgical material, were incubated with [ 3H]thymidine and the test agent. UDS was measured by quantitative autoradiography as net nuclear grains (NG). By similar methods, UDS and RDS (S-phase cells) were measured in hepatocytes isolated from rats and mice treated by gavage with TMP (500 mg/kg) or UG (100 to 5,000 mg/kg). A dose-related increase in UDS activity was observed in rat hepatocytes treated in vitro with 0.05 to 0.10% (v/v) UG. These doses were, however, toxic in both mouse and human hepatocyte cultures. Weak UDS activity was observed in hepatocytes isolated from male and female mice treated 12 hr previously with UG. No UDS was induced in rat hepatocytes treated in vivo or in vitro with TMP. Twenty- and fourfold increases in the percentage of cells in S-phase were observed 24 hr after treatment with TMP in male and female mice, respectively, as compared to a fivefold increase in male rats. UG increased the percentage of S-phase cells in male mice by ninefold but failed to induce RDS in females. Thus, there appears to be genotoxic compounds in UG that can be detected in cultured hepatocytes and in the livers of exposed mice. The lack of UDS activity in rat liver was consistent with the reported lack of liver tumors in chronically exposed rats. However, neither the UDS nor the RDS responses in mice exposed by gavage correlated to the sex-specific pattern of liver tumors observed in the 2-year bioassay.

Microinjection of partially purified protein factor restores DNA damage specifically in group A of xeroderma pigmentosum cells.

Microinjection of cell extracts prepared from both human placenta and HeLa cells into xeroderma pigmentosum (XP) cells of complementation group A restores unscheduled DNA synthesis (UDS) in these cells after UV irradiation [de Jonge, A., Vermeulen, W., Klein, B. & Hoeijmakers, J. (1983) EMBO J. 2, 637-641]. These cells also showed normal resistance to UV irradiation. The half-life of the factors in the cell extracts corresponding to the UDS activity (factor A) was 14 hr in XP cells of group A, and the maximal level of UDS was exerted 2 hr after microinjection. The factors were sensitive to protease treatment but not to RNase treatment and were found to be approximately equal to 160 and approximately equal to 90 kDa by gel filtration. These two fractions of the factor(s) acted specifically in XP cells of complementation group A among complementation groups A, B, C, D, F, G, and probably E and H.

Assessment of genotoxic potential of ethylenediamine: in vitro and in vivo studies

Ethylenediamine (EDA) was evaluated for potential genotoxic activity using a battery in vitro and in vivo mammalian tests. The tests employed were the Chinese hamster ovary (CHO) gene mutation assay, the sister-chromatid exchange (SCE) test with CHO cells, unscheduled DNA synthesis (UDS) assays with primary rat hepatocytes and a dominant lethal study with Fischer 344 rats. EDA did not produce a positive, dose-related, mutagenic effect in either the CHO mutation assay or in the SCE test when evaluated both with and without the addition of a rat-liver S9 activation system. With hepatocytes, no positive effects of EDA upon UDS values were noted in 2 separate studies using either a scintillation counting procedure or an autoradiographic method to determine UDS activity. In a dominant lethal study, male rats fed for 23 weeks with dietary levels of EDA · 2HC1 of 0, 0.05, 0.15 or 0.50 g/kg/day, and mated with 1 virgin female/week for 3 consecutive weeks, showed no dose-related or statistically significant effects upon fertility, total number of implantations/female, or the number of living and dead implants per female; marked effects upon the incidence of dominant lethal mutations were noted in the positive control group injected intraperitoneally with one dose of 0.25 mg/kg triethylenemelamine. We conclude that EDA was not genotoxic in the in vitro and in vivo mammalian test systems employed.

Drosophila mutations at the mei-9 and mus(2)201 loci which block excision of thymine dimers also block induction of unscheduled DNA synthesis by methyl methanesulfonate, ethyl methanesulfonate, N-methyl- N-nitrosourea, UV light and X-rays

The mei-9 and mus(2)201 mutants of Drosophila melanogaster were identified as mutagen-sensitive mutants on the basis of larval hypersensitivity to methyl methanesulfonate and characterized as excision repair-deficient on the basis of a greatly reduced capacity to excise thymine dimers from cellular DNA. The high degree of larval cytotoxicity observed with a variety of other chemical and physical agents indicated that these mutants may be unable to excise other important classes of DNA adducts. We have measured the ability of the single mutants and the double mutant combination mei-9;mus(2)201 to perform the resynthesis step in excision repair by means of an autoradiographic analysis of unscheduled DNA synthesis (UDS) induced in a mixed population of primary cells in culture. The 3 strains exhibit no detectable UDS activity in response to applied doses of 1.5–6.0 mM methyl methanesulfonate, 1.0–4.5 mM N-methyl- N-nitrosourea or 10–40 J/m 2 254-nm UV light, dose ranges in which control cells exhibit a strong dose-dependent UDS response. The mei-9 and mei-9;mus(2)201 mutants also have no detectable UDS response to X-ray doses of 300–1800 rad, whereas the mus(2)201 mutant exhibits a reduced, but dose-dependent, response over this range. These data correlated well with the degree of larval hypersensitivity of the strains and suggest that mutations at both loci block the excision repair of a wide variety of DNA damage prior to the resynthesis step.