Further examination of the effects of nitrosylation on Alternaria alternata mycotoxin mutagenicity in vitro.

Previously, Alternaria extract and metabolite mutagenicities+/-nitrosylation were characterized using Ames Salmonella strains TA98 and TA100, which are both reverted at GC sites. To examine other targets for mutation, the metabolites Altertoxin I (ATX I), Altenuene (ALT), Alternariol (AOH), Alternariol monomethyl ether (AME), Tentoxin (TENT), Tenuazonic acid (TA) and Radicinin (RAD) were reexamined+/-nitrosylation, using Ames Salmonella strain TA97, sensitive to frameshift mutations at a run of C's, as well as strains TA102 and TA104, reverted by base pair mutations at AT sites and more sensitive to oxidative damage. ATX I was also assessed for mammalian mutagenicity at the Hprt gene locus in Chinese hamster V79 lung fibroblasts and rat hepatoma H4IIE cells. When tested from 1 to 100 microg/plate without nitrosylation, ATX I was mutagenic in TA102+/-rat liver S9 for activation and weakly mutagenic in TA104+/-S9, demonstrating direct-acting AT base pair mutagenicity. AOH was also directly mutagenic at AT sites in TA102+/-S9 while AME was weakly mutagenic in TA102+/-S9 and TA104+S9. Nitrosylation of ATX I enhanced mutagenicity at AT sites in TA104+/-S9 but produced little change in TA102+/-S9 compared to native ATX I. However, nitrosylated ATX I generated a potent direct-acting frameshift mutagen at C sites in TA97+/-S9. While ATX I was not mutagenic in either V79 cells or H4IIE cells, 5 and 10 microg/ml nitrosylated ATX I produced a doubling of 6-thioguanine resistant V79 colonies and 0.5 and 1 microg/ml were mutagenic to H4IIE cells, becoming toxic at higher concentrations. These results suggest ATX I, AME and AOH induce mutations at AT sites, possibly through oxidative damage, with nitrosylation enhancing ATX I frameshift mutagenicity at runs of C's. Nitrosylated ATX I was also directly mutagenic in mammalian test systems.

Characterization of protein interactions with positive and negative elements regulating the apoVLDLII gene.

Synthesis of avian apo very-low-density lipoprotein (apoVLDL)II is estrogen dependent and liver specific. Competence to express the apoVLDLII gene is not acquired until days 7-9 of embryogenesis and thus lags 5-6 days behind appearance of the liver primordial bud. It is not known whether the delayed ability to activate the gene is attributable to hepatic estrogen receptor profiles, or a requirement for other transcription factors not expressed at earlier stages of embryogenesis. The latter possibility is supported by developmental alterations in nuclease hypersensitivity flanking the gene that occur independently of estrogen administration. We have examined the influence of these hypersensitive regions on expression from the apoVLDLII promoter and have characterized novel protein-DNA interactions at two of them. One is located in a copy of the CR1 family of middle repetitive elements approximately 3.0 kb upstream from the start of the gene. We demonstrate by DNase I footprinting that the site contains an element which matches a predicted consensus silencer sequence. The other site contains no previously identified binding motifs. It is located between nucleotides -228 and -245 and is adjacent to an imperfect estrogen response element (ERE) that we demonstrate acts additively with a canonical ERE 30 nucleotides downstream. We have identified ubiquitous and liver-specific factors that display overlapping DNA contacts with the site. Mutation of G residues contacted by these proteins decreases hormone-inducible expression from the promoter 5- to 8-fold. Hepatic levels of the liver-enriched factor interacting with this site increase abruptly between days 7 and 9 of embryogenesis, suggesting that it may be an important determinant of the ability to express the apoVLDLII and possibly other liver-specific genes.

Characterization of liver-enriched proteins binding to a developmentally demethylated site flanking the avian apoVLDLII gene.

Although the avian apoVLDLII gene is normally expressed exclusively in the liver of the laying hen, the gene can be activated by estrogen in birds of either sex beginning between days 7-9 of embryogenesis. Developmentally programmed demethylation of sites in the 5'- and 3'-flanking regions of the gene have been shown to occur during this period of embryogenesis, suggesting that they may reflect changes in protein-DNA interactions that are involved in the acquisition of competence to activate the apoVLDLII gene. We have detected specific protein interactions at one location approximately 2.6 kb upstream from the apoVLDLII gene, that includes an Msp I site whose methylation status changes between days 7 and 9 of embryogenesis. The sequence of this region bears significant similarity to binding sites of members of the bZIP family of liver-enriched or -specific factors such as C/EBP, DBP, and LAP, that are characteristically produced relatively late during liver development. In the studies described here, we demonstrate that proteins binding to the upstream apoVLDLII site do not correspond to previously identified liver-enriched or -specific factors. They also display a pattern of activity during development and in human and avian hepatoma cell lines indicating that their expression is increased in proliferating cells. Southwestern blotting and UV cross-linking studies indicate that two proteins of approximately 60 kD are capable of binding to the site and we describe the purification of these factors from crude nuclear protein extracts obtained from rooster liver.

Examination of selected food additives and organochlorine food contaminants for androgenic activity in vitro.

In order to produce a reporter gene assay for androgenic chemicals, a constitutive expression vector coding for the human androgen receptor and a reporter construct containing the firefly luciferase coding sequence under transcriptional control of the androgen responsive MMTV promoter were cotransfected into the androgen-insensitive human PC-3 prostate carcinoma cell line and stable transfectants selected. One colony of transfectants, PC-3 LUCAR+, was characterized further. 5alpha-Dihydrotestosterone (DHT) enhanced luciferase activity in a linear fashion for up to 3 days of culture. The Kd for DHT activation was within the range of 25.0-60.0 pM (r2 values >0.95). Flutamide competitively inhibited DHT activation (mean Ki value of 0.89 microM). Progesterone, estradiol, dexamethasone, and hydrocortisone were weak agonists (100-fold less effective than DHT) and diethylstilbestrol was without effect. The effects of organochlorine food contaminants (0, 0.1, 1.0, and 10.0 microM) on luciferase activity in PC-3 LUCAR+ cells were determined after exposure to the chemical for 18 h in the presence and absence of DHT (50 pM). 1,1-dichloro-2,2-bis(p-chlorophenyl)-ethylene (p,p'-DDE) induced luciferase activity in the absence of DHT (100 microM p,p'-DDE equivalent to 50 pM DHT), but in the presence of DHT (50 pM), p,p'-DDE acted antagonistically. 2,3,7,8-Tetrachlorodibenzo-p-dioxin, kepone, butylated hydroxyanisole, and butylated hydroxytoluene all partially inhibited activation by DHT (50 pM) but alone had little or no effect. Toxaphene at 10 microM induced luciferase activity in the absence of DHT but decreased cell viability. Alpha- and delta-Hexachlorocyclohexanes (HCH) at 10 microM antagonized the DHT effect, but beta-HCH and gamma-HCH mirex, photomirex, oxychlordane, cis- and trans-nonachlor were without effect. Thus, of the chemicals tested, some interact with the human androgen receptor in vitro as agonists, others as antagonists, and some as partial agonists/antagonists.

Differences in nucleotide and base DNA excision repair observed during mitogenic stimulation of bovine lymphocytes.

The bromodeoxyuridine density-shift technique was used to examine nucleotide and base DNA excision repair in quiescent and lectin stimulated bovine lymphocytes damaged with either ultraviolet light or dimethyl sulfate (DMS). Compared to a number of human cell lines, quiescent lymphocytes were less proficient in the repair of both types of damage. Repair replication was enhanced upon mitogenic stimulation, but both the amount and time course of the increase in repair depended upon the damaging agent used. A 2-3-fold increase in UV light induced repair replication occurred early during stimulation and subsided only gradually as stimulation proceeded. However, the profile of DMS induced repair increased 7-fold and then decreased, in parallel with measurements of lectin-stimulated DNA replication. Estimates of average repair patch sizes showed that quiescent lymphocytes produced smaller patches of 7 nucleotides in response to DMS damage while UV light irradiation resulted in repair patches of 20 nucleotides. During stimulation, patch sizes appeared to increase to maximum values of 45 and 33 nucleotides in response to UV light and DMS, respectively, one day prior to the peak of DNA replication. These increases in patch size were followed by a gradual decrease towards unstimulated levels. However, the appearance of a DNA species of intermediate density in the gradient profiles made the interpretation of repair patch sizes in stimulated cells difficult. These results are discussed as evidence not only for differences in the mechanisms of nucleotide and base excision repair but also for changes in repair as the cell progresses through the cell cycle.

Comparison of HepG2 feeder cells generated by exposure to gamma-rays, X-rays, UV-C light or mitomycin C for ability to activate 7,12-dimethylbenz[a]anthracene in a cell-mediated Chinese hamster V79/HGPRT mutation assay.

The cell-mediated Chinese hamster V79/HGPRT mutagenicity assay is an established in vitro testing method. Although gamma-irradiated human HepG2 hepatoma cells have been used recently for chemical activation, an alternative is now needed due to scheduled retirement of the available gamma-source. X-irradiation, 254 nm UV-C light and mitomycin C were examined as possible HepG2 mitotic inhibitors, and treated cells compared for activation of 7, 12-dimethylbenz[a]anthracene (DMBA). In colony-forming assays, V79 and HepG2 cells differed in sensitivity to DMBA, with V79 survival declining sharply between 1-2.5 microM (LD50=1.75 microM) while HepG2 survival decreased gradually, beginning at 0.01 microM DMBA (LD50=0.045 microM). When HepG2 feeder cells generated by each method were included in V79/HGPRT mutation assays, activation of 1 microM DMBA was found to vary according to the mitotic inhibitor used, with mutation frequencies decreasing in the order 4000 rads gamma-rays>25 microg/ml mitomycin C>4000 rads X-rays>25 J/m2 UV-C light. Only assays containing gamma-irradiated HepG2 cells generated an increase (2-3-fold) in mutation frequency when DMBA exposure was extended from 24 to 48 h. The effect of HepG2 preincubation with either Aroclor 1254 or DMBA on feeder cell activation of DMBA was also assessed using concentrations of Aroclor 1254 (10 microg/ml) or DMBA (1.0 microM) which were found to produce optimum induction of ethoxyresorufin-O-deethylase (EROD) activity (3.1-fold and 2-fold increases, respectively). Compared to results obtained with uninduced HepG2 cells, assays incorporating HepG2 cells activated by either Aroclor 1254 or DMBA produced slightly increased V79/HGPRT mutation frequencies after 24 h of exposure to mutagen; however, a 48 h incubation with mutagen in the presence of HepG2 preincubated with either Aroclor 1254 or DMBA resulted in higher mutation frequencies regardless of the mitotic inhibitor treatment. EROD activity was also induced 1.4-fold following exposure of HepG2 cells to mitomycin C alone. Although gamma-irradiation remains the treatment of choice for producing metabolically active HepG2 feeder cells, comparison of the alternatives tested suggests that mitomycin C would be a convenient and suitable replacement.

Low levels of DNA excision repair in undamaged bovine lymphocytes.

Quiescent and concanavalin A-stimulated bovine lymphocytes were subjected to a buoyant density analysis used in excision repair studies. Despite neutral and alkaline rebands to remove replicative contamination, the CsCl gradient profiles of DNA isolated from unstimulated lymphocytes given a 6-h labelling period revealed a small amount of radioactivity in the normal-density region which is indicative of an excision repair process. It amounted to the incorporation of 8,000-20,000 molecules of thymidine per lymphocyte. In a 12-h labelling period the extent of repair incorporation was twice that measured in a 6-h period. The extent of this repair incorporation was not altered significantly during the initial 6 or 12 h of lectin stimulation when DNA-strand breaks normally present in the unstimulated cells are repaired. The same amount of repair activity was found whether the measurements were made on the same day that the lymphocytes were isolated or on the next day following an overnight incubation of the cells in culture medium. These observations indicate that lymphocytes display a spontaneous excision repair activity that proceeds continuously and at a constant rate.

In vitro investigation of toxaphene genotoxicity in S. typhimurium and Chinese hamster V79 lung fibroblasts.

The polychlorinated pesticide toxaphene has been identified as a persistent environmental contaminant and is of particular concern in the Great Lakes and Arctic regions of Canada. Inconsistencies in published in vitro genotoxicology studies have hindered risk assessments of toxaphene exposure. When toxaphene mutagenicity was re-evaluated in the Ames Salmonella/microsome assay at 10-10,000 microg/plate, a dose-dependent increase in His revertants occurred in all five strains of S. typhimurium tested (TA97, TA98, TA100, TA102 and TA104) with higher mutation frequencies observed in the absence of S9 metabolic activation. However, the mutagenic potential of toxaphene was relatively low with concentrations greater than 500 microg/plate required to induce mutation. Toxaphene genotoxicity was also examined in a mammalian system using Chinese hamster V79 lung fibroblasts with metabolic activation provided by human HepG2 hepatoma cells. Genotoxicity of 1-10 microg/ml toxaphene was examined by measuring the frequency of sister chromatid exchange (SCE) and mutation induction at the hypoxanthine guanine phosphoribosyl transferase (HGPRT) gene locus. Although small increases in SCE were observed at toxic concentrations of toxaphene approaching the LD50 (10 microg/ml), they were not found to be statistically significant relative to control. Toxaphene was also unable to induce HGPRT mutagenesis at the concentrations tested. These results show that while toxaphene is a weak, direct-acting mutagen in the Ames Salmonella Test, convincing evidence of dose-dependent SCE induction and mutagenicity at the HGPRT gene locus could not be demonstrated in V79 cells.

Formation of iodoacetic acids during cooking: interaction of iodized table salt with chlorinated drinking water.

Iodoacetic and chloroiodoacetic acids were formed when municipal chlorinated tap water was allowed to react with iodized (with potassium iodide) table salt or with potassium iodide itself. Iodoacetic acid was recently shown to be a potent cytotoxic and genotoxic agent. For analysis, samples were extracted with t-amyl methyl ether and converted to the corresponding methyl esters using methanol and sulfuric acid. The concentration of iodoacetic acid was determined by gas chromatography-mass spectrometry (GC-MS) using an authentic standard. The identities of iodoacetic and chloroiodoacetic acids were further confirmed by gas chromatography-high-resolution mass spectrometry (GC-HRMS). Certain influences of sodium hypochlorite and humic acid as well as the concentration of potassium iodide on the yields of these acids were investigated. The concentration of iodoacetic acid in tap water samples boiled with 2 g l-1 of iodized table salt was found to be in the 1.5 microg l-1 range, whilst the concentration of chloroiodoacetic acid was estimated to be three to five times lower.

3-Aminobenzamide does not increase repair patch size in mammalian cells.

Several groups of investigators have reported that the extent of repair replication following treatment of mammalian cells with dimethyl sulfate is increased by 3-aminobenzamide (3AB) over that occurring in its absence. Two plausable explanations for this phenomenon were tested. The first is that the number of nucleotides inserted per repair site, that is the repair patch size, is increased and the second is that the rate of repair is increased. Human T98G cells were treated with dimethyl sulfate and allowed to repair their DNA in the presence or absence of 3AB. It was found that the presence of 3AB did not increase the repair patch size nor the rate of removal of methylation products from the DNA, which is assumed to equal the rate of repair. The results of further experiments suggested that the 3AB effect can be explained by changes in nucleotide precursor pools.

Examination of Alternaria alternata mutagenicity and effects of nitrosylation using the Ames Salmonella test.

Molds of the genus Alternaria are common food pathogens responsible for the spoilage of fruits, vegetables, grains, and nuts. Although consumption of Alternaria alternata-contaminated foodstuffs has been implicated in an elevated incidence of esophageal carcinogenesis, the mutagenic potencies of several A. alternata toxins seem unable to account for the levels of activity found using crude mycelial extracts. In this study, the mutagenic effects of nitrosylation were examined with the major Alternaria metabolites Altenuene (ALT), Alternariol (AOH), Alternariol Monomethyl Ether (AME), Altertoxin I (ATX I), Tentoxin (TENT), Tenuazonic Acid (TA), and Radicinin (RAD) using the Ames Salmonella strains TA98 and TA100. In the absence of nitrosylation, ATX I was mutagenic when tested from 1 to 100 microg/plate in TA98 with rat liver S9 for activation, while AOH and ATX I were weakly mutagenic +/- S9 in TA100. Incubation with nitrite generally increased mutagenic potencies with ATX I strongly mutagenic +/- S9 in both TA98 and TA100, while ALT, AOH, AME, and RAD responses were enhanced in TA100 + S9. However, subsequent examination of three extracts made from A. alternata culture broth, acetone-washed mycelia, and the acetone washes showed a different mutagenic response with both broth and acetone washes directly mutagenic in TA98 and TA100 but with a reduced response + S9. The acetone-washed mycelial extract was found to have the lowest mutagenic activity of the three extracts tested. Nitrosylation had little effect on the mutagenicity of any of the extracts. Thus, while nitrosylation increases the mutagenicity of ATX I, and to a lesser extent that of several other Alternaria toxins, the results demonstrate that Alternaria produces a major mutagenic activity with a S. typhimurium response different from that found with the purified toxins. Efforts are currently underway to chemically identify this mutagenic species. Published 2001 Wiley-Liss, Inc.