In Vitro Mutagenic and Genotoxic Assessment of a Mixture of the Cyanotoxins Microcystin-LR and Cylindrospermopsin

The co-occurrence of various cyanobacterial toxins can potentially induce toxic effects different than those observed for single cyanotoxins, as interaction phenomena cannot be discarded. Moreover, mixtures are a more probable exposure scenario. However, toxicological information on the topic is still scarce. Taking into account the important role of mutagenicity and genotoxicity in the risk evaluation framework, the objective of this study was to assess the mutagenic and genotoxic potential of mixtures of two of the most relevant cyanotoxins, Microcystin-LR (MC-LR) and Cylindrospermopsin (CYN), using the battery of in vitro tests recommended by the European Food Safety Authority (EFSA) for food contaminants. Mixtures of 1:10 CYN/MC-LR (CYN concentration in the range 0.04-2.5 µg/mL) were used to perform the bacterial reverse-mutation assay (Ames test) in Salmonella typhimurium, the mammalian cell micronucleus (MN) test and the mouse lymphoma thymidine-kinase assay (MLA) on L5178YTk± cells, while Caco-2 cells were used for the standard and enzyme-modified comet assays. The exposure periods ranged between 4 and 72 h depending on the assay. The genotoxicity of the mixture was observed only in the MN test with S9 metabolic fraction, similar to the results previously reported for CYN individually. These results indicate that cyanobacterial mixtures require a specific (geno)toxicity evaluation as their effects cannot be extrapolated from those of the individual cyanotoxins.España Ministerio de Economía y Competitividad AGL2015-64558-

Short-Term Effects of Carcinogens and Irritants on the Respiratory Tract Epithelium.

Enlarged nuclei have been frequently observed as an early carcinogen-induced change in both cultured cells and in target tissues in vivo. The purpose of this project was to examine the occurence of nuclear enlargement in the upper respiratory tract of rats to provide further evidence of whether nuclear enlargement is a reliable marker of carcinogenesis, and if it could be used a short-term test for respiratory carcinogens. The first approach to this problem was to use an in vivo model. Carcinogen-induced nuclear enlargement is best demonstrated when the tissue involved has been undergoing rapid replication. Rats were thus concommitantly exposed to sulphur dioxide by inhalation (which causes a hyperplastic response in the nasal cavity) and an N-nitrosamine i. p. (nitrosopyrrolidine, nitrosomorpholine, nitrosopiperidine or nitroso-di-n-propylamine) known to cause nasal tumours in rats following systemic administration. Sections were prepared from the nasal cavity and nuclear areas were measured. There were some increases in nuclear size 24 and 72 hours after the start of treatment. This effect had been reversed 120 hours after treatment. Attempts were also made to study the effects of carcinogens in cultured respiratory epithelial cells. The culture of nasal turbinate cells (either as tissue cultures or as primary cell cultures) was unsuccessful, however tracheal rings and tracheal epithelial cells were cultured. In both these model systems the epithelial cells grew rapidly, and so no further treatment was necessary to induce a hyperplastic response. Exposure of tracheal rings to benzo(a)pyrene failed to produce any nuclear enlargement during the time period examined. This was expected as benzo(a)pyrene is not tumourigenic in rat trachea in vivo. Nitroquinoline-N-oxide and dimethylnitrosamine are both potent carcinogens in the respiratory tract and induced nuclear enlargement. A number of carcinogens and noncarcinogens were also tested for their ability to induce increases in nuclear size in primary tracheal epithelial cell cultures. The results obtained were generally in agreement with the tumourigenicity of the test chemicals. The exception to this was benzo(a)pyrene which produced a significant increase in nuclear size. This project appears to provide evidence that exposure to carcinogens can lead to increased nuclear size in the respiratory tract of rats, above that produced by a concommitant hyperplastic control, and that this may be useful as a tissue-specific shortterm test for carcinogens. The relationship between nuclear enlargement, increases in nuclear DNA content and the process of carcinogenesis is discussed

The impact of folate deficiency on the base excision repair pathway: Analysis of enzyme coordination in response to dna damage and imbalanced repair

The condition of folate deficiency has been implicated in carcinogenesis, with the strongest evidence formulated in colon cancer. The role of folate in DNA repair, DNA synthesis and methylation reactions renders this nutrient an valuable target for studying the onset and progression of cancer. Using molecular techniques to determine gene and protein expression, enzyme activity and methylation status elucidates the mechanism of DNA repair and damage in folic acid deficient animals in response to carcinogen. The findings presented in this study indicate failure to remove and repair damage in the condition of folate deficiency and suggest that the accumulation of DNA base lesions and damage can result in clonogenic selection of initiated cells, driving carcinogenesis

Mutagenesis Occuring Following Infection with Herpes Simplex Virus and the Contribution of Virus Ribonucleotide Reductase

Herpes simplex virus type 2 (HSV-2) has been associated with cervical cancer for many years and certain regions of both the HSV-1 and the HSV-2 genome can induce transformation of tissue culture cells. However, no virus transforming protein has yet been identified and no region of viral DNA appears to be stably retained in transformed cells or tumours. This implies that continued expression of a virus protein is not required and has resulted in the proposal that HSV transforms cells by a "hit and run" mechanism. HSV infection can generate chromosomal breaks together with amplification and rearrangement of cellular genes. Additionally, infection of permissive cells with UV-inactivated HSV-1 and of non-permissive cells with HSV-2 leads to an increase in the mutation frequency of the cellular hypoxanthine-guanine phosphoribosyltransferase gene. HSV-1 was also found to increase the mutagenic frequency of a plasmid-based gene located on the shuttle vector pZ189. These mutagenic effects resemble changes produced by chemical carcinogens and may represent a mechanism for transformation which would not require the retention of viral sequences. The cause of mutations occurring in HSV-infected cells is unknown but could be connected with the destabilization of doxyribonucleoside triphosphate (dNTP) pools seen in infected cells as studies of both prokaryotic and eukaryoti systems have stressed the importance of dNTP concentrations in mediating base substitutions and misincorporations. The HSV enzyme ribonucleotide reductase has a potential role in the destabilization of cellular dNTP pools since, unlike it cellular counterpart, it is not allosterically regulated. This is supported by results which show that a mutant cellular enzyme, which was deficient in allosteric control, raised the cellular mutation rate in transfected cells. In order to study the effect of HSV-2 ribonucleotide reductase on mutagenesis, expression vectors were constructed which contained the large (RRl) and small (RR2) subunits of the HSV-2 ribonucleotide reductase under the control of the inducible mouse metallothionein promoter, which responds at the transcriptional level to heavy metals such as zinc and cadmium. These constructs were used in mutagenesis assays which involved monitoring the mutation frequencies of the cellular adenine phosphoribosyltransferase gene (aprt) in Chinese hamster ovary (CHO) cells and of the plasmid-based suppressor tRNA gene (supF) in human cells. Increasing the amount of enzyme expression by zinc-induction did not increase the mutagenesis of the marker genes in either assay suggesting that virus ribonucleotide reductase expression may not be mutagenic in HSV-infected cells. Experiments using the HSV-1 mutant viruses tsl207 and tsl222, which are temperature sensitive (ts) for RRl and RR2 respectively, support this finding since these viruses were as mutagenic as wild type HSV-1 at both the permissive (P) and the non-permissive (NPT) temperatures. Although expression of the viral ribonucleotide reductase does not appear to be the cause of increased mutagenesis in infected cells, the possibility that enzyme activity is capable of producing a mutagenic effect cannot be ruled out. The mutagenesis assays described above involved only the transient expression of the viral enzyme and the lack of any mutagenic effect might be due to inefficeincies in the transfection procedure and/or insufficient expression of the viral genes. The cellular enzyme would also be present and might counteract dNTP pool imbalances brought about by viral ribonucleotide reductase expression. To address these problems, NIH3T3 and CHO cell lines were constructed using HSV-2 ribonucleotide reductase expressing plasmids that contained the G418-resistance gene (neoR). Mutations causing morphological transformation were assayed in NIH3T3 cells while in CHO cells the aprt gene was assayed. None of the G418-resistant cell lines tested were found to express RRl or RR2 and further lines will be screened for expression before repeating the experiment. The ribonucleotide reductase constructs were used to generate transgenic mice to determine the effect of enzyme expression in an animal model. The mice did not appear to be affected by the presence of a large number (50+) of viral ribonucleotide reductase sequences. Viral ribonucleotide reductase RNA was detected in these animals, however, it did not appear to be inducible and protein could not be detected. Both HSV-1 and HSV-2 increase the spontaneous mutation frequency of the plasmid based supF gene by around 6-fold which is in agreement with values obtained by others in similar studies

Oncogenetics of Lung Cancer Induced by Environmental Carcinogens

The molecular landscape of non-tobacco-induced primary lung tumors displays specific oncogenetic features. The etiology of these tumors has been largely associated with exposure to well-established environmental lung carcinogens such as radon, arsenic, and asbestos. Environmental carcinogens can induce specific genetic and epigenetic alterations in lung tissue, leading to aberrant function of lung cancer oncogenes and tumor suppressor genes. These molecular events result in the disruption of key cellular mechanisms, such as protection against oxidative stress and DNA damage-repair, which promotes tumor development and progression. This chapter provides a comprehensive discussion of the specific carcinogenic mechanisms associated with exposure to radon, arsenic, and asbestos. It also summarizes the main protein-coding and non-coding genes affected by exposure to these environmental agents, and the underlying molecular mechanisms promoting their deregulation in lung cancer. Finally, the chapter examines the anticipated challenges in personalized intervention strategies in non-tobacco-induced lung cancer

Investigations of radiation-induced and spontaneous chromosomal inversion formation and characteristics

2014 Fall.To view the abstract, please see the full text of the document

An investigation of BPDE-DNA adducts in t-lymphocytes from progeny mice exposed to benzo(a)pyrene during midgestation, 1996

Immune deficiency induced by benzo(a)pyrene (BP) in mice has been attributed to the metabolic intermediate benzopyrene-7,8-dihydrodiol-9,10-epoxide (BPDE). Our studies on mice progeny immune status after exposure of the pregnant female at midgestation (11-12 days) show a decided suppression in T-cell mediated immunity (CMI), reflected by deficiencies, particularly in the T-cell subsets L3T4 and Lyt 1. We hypothesize that this condition could also be a result of covalent binding of BPDE to DNA in T-lymphocytes (i.e., formation of BPDE-DNA adducts), which affect their functional status. New Zealand white rabbits were immunized with BPDE-DNA that was synthesized in vitro and conjugated to a strong immunogen, BSA, for the production of antibody against BPDE-DNA, confirmed by ELISA. Immunoaffinity purified anti-BPDE-DNA detected BPDE-DNA adducts in T-lymphocytes by ELISA and flow cytometric analysis. Finally, adduct positive cells were determined to have function when CM I was measured by the mixed response assay