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Thesis (Ph. D.)--West Virginia University, 2002. / Title from document title page. Document formatted into pages; contains x, 140 p. : ill. (some col.). Includes abstract. Includes bibliographical references (p. 120-131).

Pyrrolizidine alkaloids (PAs) produce a variety adverse hepatic effects, including acute toxicity, carcinogenicity and potent, persistent antimitotic effects. Additionally several have been evaluated as antineoplastic agents. PAs constitute significant health hazards to man and domestic animals. The mechanism(s) by which PAs induce these effects are not known. These studies were designed to test the hypothesis that some or all of the adverse hepatic effects and possibly the antineoplastic activity of PAs associate with or are mediated by a genotoxic interaction with cellular DNA. The first objective of the studies was to verify the in vivo gentoxicity of the PAs, in the process characterizing the type(s) of DNA damage induced. Hepatic DNA damage induced by the model PA monocrotaline (MCT) was assessed following i.p. administration to adult male Sprague-Dawley rats. DNA damage was characterized by the alkaline elution technique. MCT was found to induce both DNA-DNA interstrand and DNA-protein cross-links. No evidence was seen for the induction of DNA single-strand breaks, although the presence of small numbers of DNA single-strand breaks could have been masked by the overwhelming predominance of DNA cross-links. DNA-DNA interstrand cross-linking reached a maximum within 12 hr and thereafter decreased over a protracted period. By 96 hr post administration, the calculated cross-linking factor was no longer statistically different from zero (control). Further studies were performed to test the effects of agents known to modulate the formation/disposition of the proposed reactive intermediate and the toxic effects of the PAs. Consistent with its involvement in the mechanism of the toxicity of the PAs, genotoxicity was shown to modulate in the same direction and to similar degree as does the toxicity. Other PAs, or derivatives thereof, were evaluated in addition to MCT. Structural requirements for DNA cross-linking potential were shown to be similar to those required for the induction of toxic and antimitotic effects, again consistent with the involvement of DNA cross-linking in the mechanism of these effects. Indicine N-oxide however, an experimental antineoplastic agent, was shown not to mediate its cytotoxic effects via this mechanism.

Genotoxicology involves the assessment of a substance’s ability to induce DNA damage after exposure to humans. DNA damage is an underlying cause of mutations that are likely to initiate carcinogenesis. Furthermore, the investigation of low dose responses in genotoxicology testing helps to improve health risk assessment by establishing whether DNA reactive compounds follow linear or non-linear (thresholded) dose response relationships. The current assumption for direct acting genotoxins is that the relationship between exposure to genotoxic chemicals, DNA damage formation and the induction of mutagenic changes is linear. However, it is known that mutations are not produced directly by DNA adducts as DNA repair activity limits the proportion of adducts processed into mutations. It is therefore possible, that no observed effect levels (NOEL) may exist for some genotoxins. The main aim of this thesis was to improve in vitro genotoxicity testing by assessing the low dose response relationships for the genotoxic agents mitomycin-C (MMC), 4-nitroquinoline 1-oxide (4NQO) and cytosine arabinoside (araC). Furthermore, the automated micronucleus slide scoring system Metafer was validated and used for these studies. In addition, the mechanism of action of each test component was further investigated by follow up experiments to gain a better understanding of the processes involved in this type of damage. The in vitro micronucleus assay for the detection of chromosomal damage revealed non­linear dose response relationships following low dose exposure of MMC and araC, while 4NQO revealed a weak clastogenic potential. The semi-automated scoring protocol for the Metafer-System proved to be a rapid and accurate system for scoring micronuclei. DNA repair plays most likely a major role in these non-linear responses by removing genetic damage induced at low levels. Furthermore, p53 was shown to be involved in the DNA damage response in human lymphoblastoid cells, through cell cycle delay and the induction of apoptosis. In addition, this work confirmed that a proper dosing regime, accurate toxicity measurements and the appropriate choice of cell type are cmcial criteria for defining the dose response relationships and the induction of genotoxicity and cytotoxicity.

ro-carcinogens e.g. B[a]P and PhIP require metabolic activation to exert genotoxicity. Both B[a]P and PhIP are known to cause different types of cancers, however, very little is known about the dose response of these two chemicals at low concentrations. This study was conducted to determine the effect of low doses of B[a]P and PhIP and their exposure time on cell lines with varying levels of metabolic activity. Micronucleus and HPRT assays were conducted to determine the effect of low doses of B[a]P on micronuclei induction and mutation frequency following 4 or 24 h exposure. MCL-5 and HepG2 cell lines showed higher induction of micronuclei irrespective of B[a]P dose and exposure time. Micronuclei induction was least in AHH-1 while TK-6 cells showed no micronuclei induction. HPRT assay also showed higher mutation frequency in MCL-5 as compared to AHH-1 at both time exposures. Analysis of mutation spectra of MCL-5 and AHH-1 HPRT mutants revealed that the type of mutations observed in B[a]P treated cells were different to those observed in untreated control B[a]P-induced mutations were predominantly G → T transversions. Real time PCR assays revealed higher induction of CYP1A1 and CY1A2 enzymes in response to B[a]P in MCL-5 and HepG2 cell lines. Studies on PhIP showed significantly higher cytotoxicity, genotoxicity and mutation frequency in the MCL-5 and HepG2 cell lines than AHH-1 cells. Micronucleus assays (24h) revealed 1.56 and 1.9-.fold increase in micronuclei induction in MCL-5 and HepG2, respectively as compared to control. A similar trend was observed in 4h PhIP exposure study, where MCL-5 and HepG2 had 1.83 and 1.92-.fold increase respectively. These findings are in line with the metabolic potential of the cell lines. Real-time PCR assays showed that over all, expression of CYP1A1 and CY1A2 was higher in HepG2 than MCL-5 following PhIP exposure for 24h. PhIP was observed to induce a significantly higher mutation frequency in MCL-5 cell lines than untreated control. Mutation type also varied among PhIP treated and untreated control of MCL-5. PhIP treated MCL-5 cells showed predominantly G → T transversions. These studies showed that cells with higher metabolic activity are relatively more capable of activating B[a]P and PhIP and therefore show higher genotoxicity in response to dose and exposure of these pro-carcinogens. Considering the results of this study, potential risk of B[a]P and PhIP induced cancers has been discussed.

This thesis presents a study on possible genetic consequences of the exposure to the space environment during space missions The present study was undertaken in co-operation with the Canadian Space Agency, and involved the analysis of the lymphocyte samples taken from experienced cosmonauts and trainees. For the analysis of genotoxicity of the space environment, a T-lymphocyte hprt clonal assay has been employed. In order to distinguish between artefacts associated with this method and the spaceflight-related effects, we have conducted a series of in vitro reconstruction experiments. In these experiments we have analysed interactions between plating efficiency (PE) of T-lymphocytes and efficiency of mutant recovery. Using 12 pairs of independent wild type (WT) and mutant clones, we have demonstrated an inverse correlation between initial viability of the WT cells and survival of mutant cells (r = 0.3496, p < 0.05). Our data suggest that the presence of WT cells in the selection plates does suppress the recovery of mutants in HPRT assay. This effect is stronger in samples with high PE, and may be a source of large error in estimation of mutant frequencies (approx. 3-fold in the range of PEs from 10% to 60%), which is especially relevant when samples with different PEs are compared. Analysis of samples from cosmonauts was conducted in two experiments. The first experiment involved 5 samples taken in 1992 from cosmonauts who have completed spaceflights ranging in duration from 7 to 365 days. Hprt mutant frequencies (MF) in these samples were 2.5–5 times higher than the age-corrected values for healthy, unexposed subjects in Western countries (Tates et al., 1991; Branda et al., 1993), and 2-3-fold higher than those determined for unexposed individuals residing in Russia (Jones et al., 1995). The cosmonaut mutational spectrum differed from that of unexposed healthy subjects (p = 0.042), and showed a higher incidence of splicing errors, frameshifts, and complex mutations. Distribution of base substitutions was remarkably similar to that observed in Russian twins sampled at the same period (Curry et al., 1998), thus suggestive of possible environmental, diet, or life-style related exposures. The second study was conducted on samples taken 5 years later and involved trainees and a group of cosmonauts with more uniform (at least 6 months) and recent flight experience. Hprt MFs in both cosmonaut and trainee groups were virtually identical (17.2 ± 0.6 and 17.6 ± 4.7 × 10⁻⁶ respectively), and approximately 2-fold higher than in matching Western controls, although considerably lower than in our previous observations. Mutational spectra in both datasets were very similar to that observed in our earlier study, and were significantly different from spontaneous data (p = 0.031–0.038). Distribution of base substitutions, however, did not show any differences. Our data indicate that the space environment is not genotoxic at the hprt locus. At the same time, uniformly high MFs observed in all studied groups suggest that the level of the mutagenic burden in at least megalopolis areas of Russia may be considerably larger than in the West. Also, there are some indications of a possible restructuring of mutagenic burden in post-transitional Russia. / Graduate

1,2,3-Trichloropropane (TCP) causes rat hepatic DNA damage in the form of DNA single strand breaks. This damage was dose and time dependent. In vivo ¹⁴C-TCP equivalents covalently bound to hepatic protein, RNA and DNA. Glutathione depletion with L-buthionine-(R,S)-sulfoximine increased binding to protein by 342% while it decreased binding to DNA by 56%. The in vivo binding data suggest a dual role for glutathione in the bioactivation of TCP. In vitro rat hepatic microsomes activated TCP to species which covalently bound to microsomal protein. Rat liver microsomes also bioactivated TCP to the direct acting mutagen 1,3-dichloroacetone. 1,3-Dichloroacetone was identified as the major microsomal protein binding species through conjugation with N-acetylcysteine to form 1,3-(2-propanone)-bis-S-(N-acetylcysteine) which accounted for 87% of all TCP microsomal metabolism. These findings support a role for 1,3-dichloroacetone as a mutagenic metabolite of TCP. Carbon-13 nuclear magnetic resonance was used to identify directly the urinary metabolite of ¹³C₃-TCP (99 atom % enrichment). Urine was investigated directly using proton-decoupled ¹³C and two-dimensional homonuclear correlated nuclear magnetic resonance spectroscopy. Spectral shifts have been assigned to N-acetyl-S-(2-hydroxy-3-chloropropyl)cysteine, 1,3-(2-propanol)-bis-S-(N-acetylcysteine), N-acetyl-S-(2-hydroxy-2-carboxyethyl)cysteine, 2,3-dichloropropionic acid, 2-chloroethanol, ethylene glycol and oxalic acid by comparison to spectra of authentic standards. No unchanged TCP was detected. From the results obtained it is concluded that metabolism of TCP by cytochromes P450 and by glutathione conjugation can result in the formation of reactive metabolites of TCP which may be responsible for TCP genotoxicity.

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Orientador: Eliana Aparecida Varanda / Banca: Elisângela de Paula Silveira Lacerda / Banca: Lusânia Maria Greggi Antunes / Resumo: Os ensaios de toxicologia genética são utilizados como base no desenvolvimento de novos fármacos, uma vez que podem identificar rapidamente agentes que causam danos ao material genético. Agências regulatórias preconizam a utilização de ensaios que identificam danos genotóxicos, de forma que possam servir como triagem de processos que levam à carcinogênese. Na busca por novos fármacos, a química inorgânica medicinal representa um campo de grande promessa, com potencial de expansão devido a diversidade química e reatividade dos metais. Especialmente, os complexos de rutênio, têm recebido destaque no tratamento de doenças como o câncer e a tuberculose multi-droga resistente (TB-MDR). Neste contexto, este trabalho teve como objetivo, avaliar os efeitos toxicogenéticos e antitumorais de três complexos de rutênio(II), com promissora atividade anti-TB-MDR, denominados de SCAR 4, SCAR 5 e SCAR 6 e, diante da importância do conhecimento farmacocinético de novos fármacos, outro objetivo foi avaliar a permeabilidade in vitro desses complexos. A avaliação toxicogenética foi realizada por meio dos ensaios de mutação gênica reversa com Salmonella typhimurium (Teste de Ames) e pelo ensaio do micronúcleo citoma com bloqueio da citocinese (CBMN-cit) em ausência e em presença do sistema de metabolização. Ensaios de sobrevivência clonogênica foram utilizados para avaliar a viabilidade das células CHO-K1 e HepG2, utilizadas no ensaio do CBMN-cit. A avaliação da atividade antitumoral foi investigada por meio de ensaios de citotoxicidade frente às linhagens tumorais humanas Caco-2, DU-145, HeLa, HepG2 e MDA-MB-231, pela capacidade de inibição da topoisomerase I humana (Top IB) e por ensaios físico-químicos de interação com o calf thymus DNA (ct-DNA). A permeabilidade foi analisada por meio do ensaio in vitro de permeação em monocamadas de células Caco-2 na presença e ausência de albumina do soro... / Abstract: The genetic toxicology assays are used as the basis in the development of new drugs since it's may rapidly identify agents that cause damage to the genetic material. Regulatory agencies have recommend the use of assays that identify genotoxic effcts, thus they may serve as a screening processes that lead to carcinogenesis. In the search of new molecules for therapeutic purposes, medicinal inorganic chemistry is a major promise of the field, with potential expansion due to chemical diversity and reactivity of metals. Especially, ruthenium complexes, have been highlighted in the treatment of growing diseases such as cancer and multi-drug resistant tuberculosis (MDR-TB). In this context, this study aimed to evaluate the toxicogenetic effects of three ruthenium(II) complex, that showed promising anti-MDR-TB activity, called SCAR 4, SCAR 5 and SCAR 6. In addition to this safety profile, and cosidering the traditionally antitumor activity of ruthenium complexes, this study also aimed to evaluate the antitumor activity of these complexes. In front of the importance of pharmacokinetic knowledge of new drugs, another aim of this study was to evaluate the in vitro permeability of these complexes. The toxicogenetics evaluation was performed by reverse gene mutation assays with Salmonella typhimurium (Ames test) and by Cytokinesis-block micronucleus cytome assay (CBMN-cyt). In both assays, we used models to assess the effect of complexes's metabolization. The clonogenic survival assays were used to assess the viability of HepG2 and CHO-K1 cells, that it has been used in CBMN-cyt assay. The evaluation of the antitumor activity was investigated by means of cytotoxicity assays with the human tumor cell lines Caco-2, DU-145, HeLa, HepG2 and MDAMB-231, by the ability to inhibit human topoisomerase I (Top IB) and physico-chemical assays of interaction with the thymus calf DNA (ct-DNA). The permeability was examined by means of in vitro ... / Mestre

Graduation date: 1999