Arsenic toxicity is species-dependent for mammals and is transformed to other metabolites in various organs. In order to understand its metabolism, <i>in-vitro</i> simulation experiments were set up for various organ tissues. As(V) was transformed to As(III), while DMA(V) was changed to DMAS in sheep rumen, but the suspected MA-V) was not formed. This is different for the incubation of seaweed in rumen fluid, which points to a different unknown source for MA(V). In another study, dog MDCK kidney cells showed that inorganic arsenicals were methylated in kidney cells while the exposed DMAS was only oxidized to DMA(V) in kidney cells, and the exposed DMA(V) rarely transformed. The toxicities of arsenicals in dog kidney cells is dependant upon the uptake rates and the transformation of arsenicals. DMAS was as toxic as the inorganic arsenic species and an order to magnitude more toxic than the oxo-species. Methylated arsenic species can accumulate in hair and wool and can be used as a biomarker for arsenic ingestion. When methylated compounds have different arsenic species as precursor material, such as arsenosugars, more DMA(V) and DMAS are found in keratinous tissues. Here, the horn and wool from the seaweed-eating sheep were used as a study object. Sheep horn can be used for monitoring arsenic exposure. The penetration of arsenic through human skin showed that As(III) and DMA(V) penetrate the quickest through skin while only As(III) and As(V) accumulated in skin. As(III) and DMA(V) were 40 times faster at penetration than As(V) and arsenosugars. The incubation of exposure matrix, for example seaweed extract, can protect arsenic penetration when DMA(V) or arsenosugars are being investigated, but it does not work well in As(III) contaminated seaweed extract.

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No description available.

There is a constant need to determine the genotoxic potential of the agents to which the human population is exposed. The stringent testing of new products is legislatively controlled and dependent on the accumulation of sufficient scientific data to allow an analysis of the risk. It is important to predetermine any risks in the workplace prior to the presentation of disease and to provide factual public information on personal exposure e.g. the risks associated with UV light. Various experimental assays have been developed to assess the genotoxicity, mutagenicity and mcarcinogenicity of given physical and chemical agents. The Polymerase Arrest- Polymerase Chain Reaction (PA-PCR) assay was employed to investigate the genotoxic effects (DNA adducts, DAN strand breaks and DNA crosslinking) of various physical and chemical agents on naked isolated DNA. The assay was modified to provide two adapted methods, which increased the sensitivity of the assay to report DNA damage at significantly lowered exposure levels. The ability of the PA-PCR assay to perform as an initial screening process for genotoxic activity was assessed and determined.

Sophisticated molecular methods for the identification of DNA damage in response to PAHs are presented in this thesis. They were used to analyse placental samples from Kuwaiti mothers exposed to this pollution during pregnancy. <SUP>32</SUP>P-postlabelling assay (PPL) has been employed to detect exposure-related DNA adducts in placental tissues from non-smoking, smoking UK mothers (negative and positive controls) and non-smoking Kuwaiti mothers exposed or unexposed to oil well fires as it represents an easily obtainable alternative tissues for human biomonitoring. It was unequivocally shown that human placental tissues from UK smoking mothers had adduct levels nearly two fold higher than levels in UK mothers who do not smoke. Analysis of placental DNA-adducts from Kuwaiti non-smoking mothers who were exposed to oil well fires showed approximately the same level of adducts in UK mothers who smoke. In order to determine whether the higher adduct levels in Kuwaiti mothers exposed to pollution during the fires was in fact due to the pollution, further analysis from Kuwaiti non-smoking mothers collected after one year of the oil well fires was undertaken. The total level of adducts in this group showed statistically no difference from that seen in individuals exposed to the pollution. Finally, the P53 mutational spectrum in placental DNA obtained from UK non-smoking, smoking and Kuwaiti non-smoking mothers exposed or unexposed to oil well fires was assessed using PCR amplification and RSM. RSM revealed seven mutations: a single transition and frameshift at codon 281-282 in exon 8 of a non-smoking mother and six transitions in codon 247-248 in exon 7 of Kuwaiti non-smoking mother exposed to oil well fires. No relationship was found between cigarette smoking and P53 mutations, and between the adduct levels and P53 mutations in Kuwaiti samples.

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The major aim of the present study was to identify groups of chemicals that were clastogenic at low and high toxicity and examine patterns of gene expression in an experimental cell line after dosing with these chemicals in order to achieve greater understanding of cellular responses to insult from these chemicals. The first part of this thesis focuses on cytogenetic identification of chemicals that are clastogenic at low and high toxicity. The micronucleus assay was chosen to compare pairs of chemicals that had the same mechanism of clastogenic action i.e. Alkylation, but different toxicity profiles. Results showed that there were two distinct groups of chemicals, those clastogenic at low and high toxicity, and those clastogenic at high toxicity only. The second part of this thesis focuses on the gene expression changes within the human cell line AHH-1 after dosing with chemicals of differing toxicity profiles. In order to examine gene expression change, cDNA arrays were used. Results showed significant change in gene expression between clastogens active at low and high toxicity. The group of chemicals clastogenic only at high toxicity had an increase in the expression of heat shock protein genes whereas the chemicals that act at low toxicity did not induce a change in heat shock protein gene expression despite dosing at up to 50% toxicity. Array results also showed a difference in DNA damage and repair signalling gene expression after insult from high and low toxicity clastogens.

Epoxybutene (EB) and diepoxybutane (DEB) are metabolites of 1,3-butadiene (BUT), a carcinogen. They both react with DNA to form monoadducts, while DEB can also form crosslinks. Here, studies on DNA adducts formed by EB and DEB are presented. The aims were to develop methods for monitoring human exposure to BUT and to assist mechanistic studies on BUT-induced carcinogenesis. EB and DEB were reacted with deoxynucleosides, deoxynucleotides, polydeoxynucleotides and calf thymus DNA. The major product of the reaction of EB with dGMP was a 7N(1-hydroxy-3-buten-yl) dGMP. The N7-guanine base was previously synthesised and characterised by MS. Two N7-EB-guanines were detected by HPLC after depurination of treated DNA from human lymphocytes exposed <i>in vitro</i>. The synthetic N7-EB-dGMP was postlabelled and analysed. The low labelling efficiency limits its use as a marker of BUT exposure if <SUP>32</SUP>P-postlabelling is used. HPLC analysis of DEB-dNps and polydNps identified an adenine (DEB-dAMP) and several guanine (DEB-dGMPs) adducts. A HPLC/postlabelling procedure for the detection of DEB-dAMP was developed and the adduct detected in poly(dA-dT) (dA-dT), calf thymus DNA and DEB treated CHO cells. The stability, the amount and the labelling efficiency of this adduct suggest it could be a suitable indicator of BUT exposure. Preliminary MS analysis of the corresponding nucleoside indicates an adenine adducted at the N6-position. Modified guanines corresponding to synthetic standards were detected in poly(dG-dC) (dG-dC). Synthetic DEB-dGMPs were postlabelled and conditions for their TLC separation established.

This thesis involved the development of a range of assay systems for the detection of environmental mutagens and carcinogens. Initially a protocol was optimised for the induction of mitotic gene conversion in stationary-phase cultures of the yeast <i>Saccharomyces cerevisiae</i>, strain JD1 following exposure to compounds which require exogenous metabolic activation, which involved an initial incubation at 37<SUP>o</SUP>C for 2 hours followed by a 16 hour incubation at 28<SUP>o</SUP>C. This protocol was found to be effective for the detection of cyclophosphamide and sterigmatocystin. In two separate studies, the activities of a total of 14 different compounds were then investigated in yeast using this, and other protocols involving exponential-phase cultures. In the first study, benzidine and diaminoterphenyl were detected, although, despite being structural analogues, their metabolic requirements differed. Dimethylaminoazobenzene and cyanodimethylaniline could not be detected under any of the conditions examined. In the second of these studies 8 carcinogens and 2 non-carcinogens were examined. Only one of the carcinogens, Acrylonitrile, was detected. The inactivity of the other 7 carcinogens was considered to be due to their ineffectiveness at inducing mitotic gene conversion. A third study indicated that respiratory status of the yeast strain used, had both quantitative and qualitative effects on the detection of sterigmatocystin, benzidine and diaminoterphenyl. Further studies were performed on two additional assays, chromosomal aberration induction and mammalian cell transformation, as these endpoints had proved very successful for detecting chemicals which were not readily detected in assays for other genetic endpoints. BZD was found to induce chromosomal abberrations in peripheral human lymphocyte cultures, in the absence of S9, which was in contrast to the activity detected in the yeast system. It was suggested that this was due to metabolic competence of the human lymphocyte cells. Studies on the stepwise transformation of Syrian hamster dermal cells, led to the suggestion of a model for the occurrence of aneuploidy events during this process, and their fixation at completion of transformation. The significance of this with respect to the observed occurrence of aneuploidy with cancer is discussed.

The human lymphoblastoid cell line AHH-1 (which expresses CYP1A1) and the metabolically competent derivative MCL-5 (which expresses CYP1A2, 2A6, 2E1, 3A4 and epoxide hydrolase), have been shown to be capable of detecting a variety of pro-genotoxins in the <I>in vitro</I> binucleate micronucleus assay. In these studies, it was possible to identify the inducibility of micronuclei by: tobacco particular matter (TPM); diesel particulate matter (DPM); "Sea Empress" slick oil; and crude oil using the binucleate micronucleus assay. A positive increase in micronuclei in mammalian binucleate cells was registered for MCL-5 cells exposed to both Slick (0.5 mg/ml) and Crude oil (0.063 mg/ml) and a positive increase in micronucleated binucleate cells was also presented in both cell lines when exposed to either DPM or TMP. These micronuclei were induced by both aneugenic and clastogenic mechanisms. Increased in mutations occurred in the <I>tk</I> mutation assay in AHH-1 for DPM (5μm/ml) and TPM (10μg/ml); in the <I>hprt</I> mutation assay AHH-1 mutations increased significantly for DPM (10μg/ml) and TPM (5μm/ml); and a significant increases in mutations was identified in MCL-5 for both assays and compounds at 5μg/ml. As the identification of micronuclei (clastogenic or aneugenic) and the production of mutations, by TPM and DPM, were shown it was appropriate to investigate whether they were due to large adducts using the <SUP>32</SUP>P-postlabelling assay and/or due to small oxidative adducts by Fapy-DNA-glycosylase. Using both restriction enzyme and sequencing analysis it was possible to identify a mutation within the p53 gene of both cell lines. However, after annexin V labelling distinguished that DNA damage induced cell death was occurring the problem of using the cell lines as a model was totally vindicated.