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Serious flaws in the scientific evaluation in the RAR incorrectly characterise the potential for a carcinogenic hazard from exposure to glyphosate. Since the RAR is the basis for the European Food Safety Agency (EFSA) conclusion, 4 it is critical that these shortcomings are corrected. [...]the WG also saw a significant increase in the incidence of pancreatic islet cell adenomas in two studies in male Sprague-Dawley rats. 14-16 In one of these rat studies, thyroid gland adenomas in females and liver adenomas in males were also increased.

Immunohistochemical methods were used to determine abundance and subnuclear distribution of DNA topoisomerase I and the Bax protein in normal and excision-repair-deficient xeroderma pigmentosum (XP) fibroblasts after irradiation of cells with gamma rays or UV light, or exposure to the topoisomerase I inhibitor topotecan. DNA topoisomerase I and Bax were monitored using antisera raised against the human proteins. In addition, topoisomerases IIalpha and IIbeta were made visible with specific antibodies. In untreated cells, DNA topoisomerase I was found to occur in the cytoplasm and in nucleoli. Irradiation with gamma rays (2-12 Gy) or UV light (0.3-1.2 mW/cm2) changed the staining pattern in nuclei such that a multitude of small topoisomerase-I-rich centers occurred, which were evenly distributed over the karyoplasm. Simultaneously nucleoli disintegrated. Treatment of fibroblasts with topotecan (6-100 microM concentrations) resulted in similar alterations although the changes were much more pronounced. Combinations of topotecan and gamma irradiation caused additive effects. We conclude that the increase in the number of topoisomerase-I-positive spots and the high fluorescence intensity of the latter may reflect three biological processes: (i) enhanced transcriptional activity (e.g. of DNA damage response genes), (ii) tagging of damaged DNA sites for repair, or (iii) initiation of apoptosis. In separate assays using normal and XP cells, a dose-dependent increase in protein reacting with Bax antibody was observed in nuclei, following treatment with gamma rays or topotecan. In addition, topotecan induced a netlike arrangement of this Bax protein in nuclei. The meshes of the net structure resembled vesicles. DNA staining with 4',6-diamidino-2-phenylindole dihydrochloride revealed that the vesicle-type structures contained DNA. Upon further incubation with topotecan, cells showing the netlike Bax arrangement eventually died. We conclude that topotecan-induced changes made visible by nuclear Bax protein are associated with apoptosis. XP cells, when treated with topotecan, responded more readily than normal cells with both an increase in nuclear Bax protein and rearrangement of Bax, indicating that UV repair functions may be required to process DNA damage inflicted by topotecan. Monitoring of DNA topoisomerases IIalpha and IIbeta in gamma-irradiated cells with antibodies revealed a dramatic increase in the IIalpha form and a redistribution of the IIbeta form representing fragmentation of nucleoli.

Tumor development is characterized by accumulation of mutations. Such mutations, if induced by carcinogens in DNA polymerase genes, would confer mutator properties on the DNA replication machinery, even at later stages of development. To investigate whether DNA polymerase delta can be mutated, we compared these enzymes from highly malignant Novikoff hepatoma cells and from regenerating normal rat liver. We sequenced the DNA polymerase delta cDNA from both sources and investigated the physico-chemical properties, inhibition characteristics, and copying fidelity of the purified enzymes. The cDNA sequences examined included the entire reading frame encoding the catalytic subunit (subunit I) of DNA polymerase delta. First-strand cDNAs were prepared from total RNA of both normal rat liver and Novikoff cells by reverse transcription, and the polymerase delta sequences were amplified by the polymerase chain reaction. cDNA (3325 bp) were sequenced. A single heterozygous mutation (CGG --> CAG) has been detected in nucleotide position 1948 (codon 648) of the polymerase delta gene from Novikoff cells, resulting in an Arg to Gln change. Position 648 lies just proximal to the conserved region VI, which is part of the \"fingers\" subdomain of alpha-like polymerases. This subdomain is involved in dNTP binding. Upon comparison of biochemical characteristics of partially purified DNA polymerase delta from both Novikoff cells and rat liver, the following properties of the enzyme from Novikoff cells were found to be altered: (i) K(50) values for nucleotide analogs (e.g. butylphenyl-dGTP) were lower, (ii) sensitivity to various antineoplastic drugs (e.g. doxorubicin, topotecan and distamycin) was enhanced, (iii) copying fidelity was decreased when primer templates containing O(6)-methylguanine were used, and (iv) the activity of DNA polymerase delta from Novikoff tumor cells was less stimulated by lactate dehydrogenase than the enzyme from normal cells. The altered biochemical characteristics of DNA polymerase delta from Novikoff cells suggest mutator properties. We conclude that the point mutation detected in the cDNA might be causally related to the observed changes in inhibition characteristics and copying fidelity.

DNA topoisomerase IIα was monitored with the monoclonal antibody Ki-S1 in human fibroblasts after irradiation of cells with γ rays from a 137Cs source or treatment with the DNA topoisomerase II inhibitor doxorubicin. DNA topoisomerase IIα was localized immunohistochemically as bright fluorescent dots in the karyoplasm. The fibroblasts investigated originated from normal human donors and a xeroderma pigmentosum (XP) patient (XP12BE). All cell lines examined showed a time- and dose-dependent increase in DNA topoisomerase IIα abundance after irradiation or treatment with doxorubicin. No principal difference in response was seen between normal and XP fibroblasts towards either treatment alone. After irradiation with 9 Gy, the effect was detectable after as little as 30 min and lasted for at least 6 h. After doxorubicin treatment, topoisomerase II overexpression occurred within less than 2 h. It passed through a maximum and began to decrease after approximately 6 h. In principle, the increase in DNA topoisomerase IIα may result from (i) architectural changes of interphase chromatin leading to enhanced accessibility of preformed enzyme to the antibody, (ii) enhanced gene expression, or (iii) enhanced stabilization of mRNA or protein molecules. The increase in enzyme levels may be part of the well-known DNA damage responses that operate in cell-protective or DNA-reparative pathways. Thus, the action of DNA topoisomerase II would serve to catalyze preparatory steps in DNA repair. We also found overexpression of the Bax protein and p16 predominantly in treated XP cells, suggesting that the DNA-damaging protocols elicited signals for apoptosis and cell-cycle arrest. From the simultaneous increase in DNA topoisomerase IIα and Bax, one may conclude that DNA topoisomerase IIα also plays role in apoptosis.

The mRNA expression of protein phosphatase inhibitor-1 (inhibitor-1) in rat liver was demonstrated using highly sensitive semi-quantitative reverse transcription polymerase chain reaction (RT-PCR). Quantification by real-time RT-PCR (LightCycler technology) yielded the same copy number of inhibitor-1 mRNA in total rat liver and isolated hepatocytes (12 copies per cell). This novel finding shows that rat liver expresses indeed inhibitor-1 mRNA, albeit in low amounts. The low copy number explains why the mRNA had not been detected by Northern blotting so far. For comparison, about 425 copies/cell were detected in brain and 2500 copies/cell in skeletal muscle from rat. The full-length coding sequence of rat liver inhibitor-1 was cloned and sequenced, 100% homology with the muscle cDNA was obtained, indicating the expression of the same gene in liver and muscle. In vitro transcription and translation yielded a protein (Mr approximately 30 kDa) which could be detected with a specific antibody by immunoblotting. This indicates an intact open reading frame of inhibitor-1 in rat liver. Immunoblotting of liver extract yielded a very weak band which comigrated with the inhibitor-1 proteins from muscle and brain. It is concluded that mRNA expression of inhibitor-1 may have implications for the regulation of protein phosphatase-1 (PP1) in rat liver.

To investigate whether or not DNA polymerases alpha, delta, and epsilon from tumor cells have acquired properties that might be responsible for mutations found in tumor development, we investigated copying fidelities of DNA polymerases alpha, delta, and epsilon from the highly malignant Novikoff hepatoma cells and compared them to the corresponding enzymes from normal rat liver. DNA polymerases were purified more than 300-fold by three chromatographic steps. Copying fidelity was studied using steady-state kinetics and an 18-mer oligonucleotide primed with a 12-mer (13-mer for extension experiments) as DNA primer-template. Three experimental approaches were chosen: i) extension of DNA primers with mismatched 3'-OH ends opposite dGMP, ii) DNA insertion of nucleotides opposite m6G in the template and iii) extension of DNA primers with mismatched 3'-OH ends opposite m6G. i) Extension of DNA primers with mismatched 3'-OH ends opposite dGMP. DNA primer templates containing G:T and G:A mispairs at the 3'-OH position of the primer were easily extended by DNA polymerases alpha, delta and epsilon from both normal rat liver and Novikoff hepatoma cells. The G:G mismatch was elongated with low efficiency. Notably, DNA polymerase alpha from Novikoff hepatoma cells extended G:A and G:G mismatches significantly faster than the enzyme from normal cells. ii) Insertion of nucleotides opposite m6G. DNA polymerases alpha, delta, and epsilon from normal rat liver preferably catalyzed incorporation of dAMP opposite m6G at dNTP concentrations < 100 microM. When dNTP concentrations were raised to > or = 100 microM, dCMP (DNA polymerases delta and epsilon) and dTMP (DNA polymerase alpha) were also incorporated. The same insertion characteristics were found for the enzymes from Novikoff cells, however, insertion efficiencies of dAMP and dCMP were significantly higher for polymerases delta and epsilon. iii) Extension of primers with mismatched 3'-OH ends opposite m6G. Only m6G:dAMP and m6G:dCMP mismatches were extended by DNA polymerases alpha, delta and epsilon from both sources. No differences in extension efficiency were observed between the enzymes from normal and hepatoma cells. Taken together, our results suggest that DNA polymerases alpha, delta, and epsilon from Novikoff cells catalyzed incorporation of the wrong nucleotides more readily and extended mismatches more easily. These results may provide a rationale why numerous mutations accumulate during tumor development.