Dimethylbenz (a) anthracene (EMBA), a carcinogen that requires metabolic activation to produce active metabolites capable of binding to DNA, has been studied in the trout and other fish. Polycyclic aromatic hydrocarbons (PAH) are of importance as they are ubiquitous in the environment and their carcinogenic effects in fish from contaminated waters are an important indication of the pollution risks to man. Since such pollution risk assessment presents the involvement of multiple agents, the study of the modulation of PAH-DNA binding produced by other agents is important. In this study the effect, of dietary pretreatment at 500 ppm, 100 ppm and 2000 ppn, using BNF, Aroclor 1254, or indole-3-carbinol (I3C) respectively on DMBA-DNA binding was examined. To study the effect of age on sensitivity to DMBA-DNA binding, adult trout and fry were used in two separate studies. The fish were fed treatment diet for at least two weeks. Fry were then injected with [³H] DMBA, at 22.4 μCi/3.9 x 10⁻² μmole/fish and adult trout at 284 μCi/1.58 μmole/fish. Liver DNA was isolated, purified and binding of radioactivity to DNA was examined and computed as the covalent binding index (CBI). Mean CBI for control dietary group vising adult trout was 1000 fold lower than for fry. Statistical analysis of covalent binding index for the treatment groups revealed that a statistically significant (p < 0.05) inhibition in DNA-DMBA binding response in adult trout and fry was produced fcy the DNF dietary treatment only. Diethylnitrosamine (DENA), a potent hepatocarcinogen in several animal species belongs also to the class of compounds that require metabolic activation. Dietary treatment and continuous exposure of trout to the carcinogen in water, have produced hepatocellular carcinctnas. The water exposure also produced a dose related DNA ethylation of the O⁶ position of guanine, believed to be the promutagenic adduct produced after DENA exposure. This study examines two other routes of exposure to DENA, in vitro hepatocyte incubations and i.p. injection. Adult trout and fry were injected with [³H] DENA. Adult fish received 3.3, 16.5, and 33 mg/kg DENA, and fry received 10, 50 and 100 mg/kg. Hepatocyte incubation was performed with doses up to 220 μM [³H] DENA, or 1 mM unlabelled DENA. DNA from fish livers and from hepatocyte pellets was isolated, purified and examined for radioactive binding of the DENA metabolites or in the case of the unlabelled DENA, was analyzed for O⁶ and N7 adduct using an HPIC technique with fluorescence detection. O⁶-EtG adduct after DENA exposure, in DNA of hepatocytes obtained from trout pretreated with beta-naphthoflavone (BNF, a known inducer of cytodhrcme P-450 dependent enzyme activities involved in the activation of xenobiotics) was below the limits of detection of the HPDC-fluorescenoe detection procedure used. To examine further if the lack of DNA binding and absence of the O⁶-EtG adduct was due to rapid repair, the persistence of O⁶-EtG after exposure to 40 nM ethylnitrosourea (ENU, a direct ethylating agent) was studied in hepatocytes at 2, 4, and 5 hours after treatment. The activity of the alkyltransferase protein involved in the repair of alkylguanines also was determined using liver extracts from adult rainbow trout. The studies did not reveal a significantly high rate of repair. It is concluded that i.p. injection and in vitro hepatocyte incubations are not a good method for studying the kinetics of activation and DNA binding of DENA in the rainbow trout. The i.p. route may lead to substantial loss of the dose of the carcinogen administered and hepatocyte incubations are limited by the toxic effects of increasing carcinogen concentration. The reasons mentioned above, coupled with low levels of metabolism of nitrosamines in trout results in the inability to detect and study the appearance, persistence and repair of the O⁶-EtG adduct. / Graduation date: 1989

Hydrocarbon solvents are widely used in the production of paints, adhesives, dyes, polymers, plastics, textiles, printing inks, agricultural products and pharmaceuticals. While the neuropathic potential of aliphatic solvents was shown in the 1970s, little is known about the neuropathic potential of aromatic solvents. The present study examines such solvents, 1,2-diethylbenzene (DEB) and its metabolite 1,2-diacetylbenzene (DAB), to determine (a) the neuropathological evidence for peripheral neuropathy in rodents treated with 1,2-DAB, (b) the neurochemical basis for the neurotoxic properties of this compound, and (c) the structural requirements for nerve fiber damage. The properties of 1,2-DAB and 2,5- hexanedione (HD) are also compared. A key finding of this thesis is that 1,2-DAB induces a 2,5-HD-like pattern of nerve damage of motor and sensory axons with focal swellings containing neurofilaments. Whereas nerve damage begins distally in 2,5-HD intoxication, with 1,2-DAB treatment axonal swellings begin intraspinally and in the proximal ventral roots of motor nerve fibers. A second key finding is the reactivity of 1,2-DAB with amino acids, notably lysine, a property that is shared with 2,5-HD. 1,2-DAB and 2,5-HD react with amino acids and proteins to form blue and yellow chromophores, respectively. Relative to 2,5-HD, 1,2-DAB is three orders of magnitude more reactive in forming high-molecular-weight species. 1,2-DAB treatment of spinal cord slices in vitro and intact sciatic nerve in vivo showed that neurofilament proteins react more readily than beta-tubulin. The heavy and medium subunits of neurofilament protein were more reactive than the light subunit. The reactivity of these four axonal proteins was in proportion to their lysine content. These data are consistent with selective accumulation of neurofilaments in giant axonal swellings. In summary, these studies have shown a relationship between the chromogenic and neuropathic properties of two gamma-diketones, one aliphatic (2,5-HD) the other aromatic (1,2-DAB). These studies are relevant to occupational and public health for at least two reasons. First, urinary chromogens generated by neuropathic aliphatic and aromatic hydrocarbons could serve as biological markers of exposure to solvents with neuropathic potential, and second, other chromogenic solvents (such as tetralin) should be considered for neuropathic potential. / Graduation date: 2002

Alpine zooplankton are exposed to a variety of stressors in their natural environment including ultraviolet radiation. Physiological coping mechanisms such as the accumulation of photoprotective compounds provide these zooplankton protection from many of these stressors. Elevated levels of carotenoid compounds such as astaxanthin have been shown to help zooplankton survive longer when exposed to ultraviolet radiation presumably due to the strong antioxidant properties of carotenoid compounds. This antioxidant capacity is important because it may ameliorate natural and anthropogenic stressor-induced oxidative stress. While previous researchers have shown carotenoid compounds impart increased resistance to ultraviolet radiation in populations of zooplankton, little work has focused on the toxicological implications of PAH induced phototoxicity on zooplankton containing high levels of carotenoid compounds. This thesis discusses research studying the role that carotenoid compounds play in reducing PAH induced phototoxicity. By sampling different lakes at elevations ranging from 9,500' to 12,700' in the front range of the Colorado Rocky Mountains, copepod populations containing different levels of carotenoid compounds were obtained. These populations were then challenged with fluoranthene and ultraviolet radiation. Results discussed include differences in survival and levels of lipid peroxidation among populations exhibiting different levels of carotenoid compounds, and the toxicological and ecological implications of these results.

The Deepwater Horizon oil spill released millions of barrels of oil into the Gulf of Mexico, impacting economically and ecologically important fishes. Polycyclic aromatic hydrocarbons (PAHs) present in the oil have been shown to cause developmental impairments in early life stage fishes, such as morphological and behavioral changes related to eye formation and visual processing following PAH exposure. Prior research reported reduced eye growth in open water, pelagic species, as well as reduced photoreceptor-specific transcription factors associated with eye development following exposure to crude oil. Though changes in transcriptomic-level pathways associated with vision and visual processing have been reported, it has yet to be determined how these changes relate to physiological or behavioral-level effects in fish. Therefore, the present studies evaluated the effect of weathered crude oil on eye development and visual function in mahi-mahi, red drum, sheepshead minnow, and zebrafish larvae. Fish were assessed through several visually-mediated behavioral assays, analyzed histologically and immunohistologically, along with subsequent transcriptomic analyses and associated gene expression changes. Larvae exposed to crude oil experienced significantly reduced abilities to exhibit optomotor or optokinetic responses relative to controls, with associated reductions in retinal development. Furthermore, genes associated with eye development and phototransduction were downregulated, with subsequent decreases in the immunofluorescence of neurological connections within the retina and a choroid-specific increase in apoptotic activity. We related oil-induced transcriptomic-level effects to morphological, physiological, and behavioral-level impairments in larval teleost fishes.