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Skin subcellular fractions, keratinocytes, living skin equivalents (LSE) and percutaneous absorption using diffusion cells were examined as in vitro models for assessment of skin xenobiotic metabolism. Cytochrome P450 monooxygenase, esterase and glutathione-S-transferase (GST) activities were investigated in rodent, pig and human skin. CYP1A1 activity was detected in rat and pig skin microsomes by ethoxyresorufin-Odeethylation. CYP2B activity was detected in rat skin microsomes by pentoxyresorufin-Odepentylation. Neither were detected in human skin microsomes. Rat keratinocytes lost cytochrome P450 activity within several hours of being isolated from skin, and were not reliably induced following exposure to B-naphthoflavone. Cytochrome P450 activities were detected in the LSE after induction by 3-methylcholanthrene. Carboxylesterase activity was detected in skin, keratinocytes, and the LSE using 4- methylumbelliferyl heptanoate as the substrate. Induction of caboxylesterase activity by 3- methylcholanthrene was shown in the LSE but not keratinocytes. GST activity was shown in skin and keratinocytes, but induction was only shown in the LSE. The ability to induce xenobiotic metabolising activity suggests that enzyme induction may be linked to cell differentiation. GST's were localised at the basal layer of the epidermis. During percutaneous absorption, DNCB was metabolised to the glutathione (GSH) conjugate, limited by the GSH available in the skin. GSH conjugation of DNCB is thought to be a detoxification pathway preventing the immune response illicited by DNCB. Studies investigating the effect of age of rat on dermal xenobiotic metabolism revealed no differences between the neonate and mature rat with respect to cytochrome P450 monooxygenase activity or esterase activity. However, neonatal rat skin showed five fold lower GST activity and three fold higher reduced GSH levels. Pig skin showed similar levels of xenobiotic metabolising activity to human skin and showed a similar metabolic profile for DNCB during percutaneous absorption, supporting its use as a better model for human than rodent skin. The LSE was a good model for studies of human dermal xenobiotic metabolism particularly with the influence of inducing agents.

Thesis.

Thesis (Ph. D.)--Ohio State University, 1986. / Includes vita. Includes bibliographical references (leaves 118-122). Available online via OhioLINK's ETD Center.

In England an estimated, 284,000 patients are admitted to NHS hospitals each year due to disease caused by smoking. It is estimated that half of all teenagers who are currently smoking will die from diseases caused by tobacco if they continue to smoke. An estimated one quarter of smokers will die after 70 years of age and one quarter before, with those dying before 70 losing on average 23 years of life. It is the addictive nature of nicotine that exacerbates the toxicities of the other components of cigarette smoke. The Royal College of Physicians has affirmed that the way in which nicotine causes addiction is similar to drugs such as heroin and cocaine. Thus studies of nicotine metabolism are of great importance since they determine the extent of which the addictive nature of nicotine can influence smoking behaviour and hence the onset of smoking related illnesses. In this area of research capillary electrophoresis (CE) is still in its infancy. But with its high resolution and high number of theoretical plates achieved, CE makes for an attractive separating device for coupling with a mass spectrometer (MS). The overall aims of this work were to produce a sensitive, highly selective, and simple CE-sample stacking/MS assay for the measurement of nicotine and its metabolites in urine, to develop a highly sensitive transient isotachophoretic/MS method, that yields detection limits comparable to that observed by HPLC/MS and with a separation efficiency to match that of CE-sample stacking/MS, and finally to characterise the metabolic activity of cytochrome P450 (e.g. CYP2D6) in the placenta, with respect to nicotine, from a representative in-vitro human trophoblast-like cell line, BeWo, via HPLC/MS and CE/UV. Analysis of urine samples was accompanied by sample clean up via SPE to ensure the appropriate removal of inorganic salts. An optimised hydrodynamic and electrokinetic injection method (HE injection) was used for CE-sample stacking/MS. HE-sample stacking/MS brought about lower detection limits (LODs of nicotine and cotinine, by CE-sample stacking/ MS (via HE injection), were found to be 0.11 and 2.25 mug/ mL, respectively) when compared to sample stacking/MS via hydrodynamic or electrokinetic injection alone. The added selectivity that the selected ion monitoring mode of MS provided ensured the clear identification of nicotine and its metabolites in urine. A counterflow transient isotachophoresis (tITP) method was developed, with MS detection, for the analysis of even lower analyte concentrations. Limits of detection for both nicotine (0.03 mug/ mL) and cotinine (0.34 mug/ mL), via HE-tlTP/MS, were considerably lower than those obtained by HE injection-sample stacking/MS, suggesting that HE-tlTP/MS could be used complementary to HE injection-sample stacking/MS. When HPLC/MS and CE/UV were used to analyse cytochrome p450 activity in the human trophoblast-like BeWo cell line, it was clear from our data that nicotine metabolism was observed. Thus it is probable that CYP mediated processes play an important role in nicotine metabolism in the placenta. When compared to HPLC/MS both HE-sample stacking/MS and HE-tlTP/MS exhibited higher resolutions and peak efficiencies; with HE-tITP/MS exhibiting comparable limits of detection and quantitation to those obtained by HPLC/MS with respect to nicotine, but not cotinine. The major improvements to the coaxial interface performance and sensitivity enhancements, has made CE/MS an attractive alternative to HPLC/MS for the determination of nicotine and its' metabolites from biological matrices.

Aspects of the toxicology of bracken (Pteridium aguilinum) were studied in sub-acute and chronic studies using male Wistar-albino rats. In a chronic study, bracken induced ileal adenomas in an 8.5 month period but after 24 months, no tumours were seen in both the quercetin- and shikimate-fed rats. Tissue ornithine decarboxylase (ODC) levels were enhanced over a 90-day test period when shikimate was fed alone or in conjunction with BBN and MNNG as initiators and with cyclophosphamide, saccharin and lithocholate as promoters, suggesting that shikimate might be a tumour promoter. Quercetin, was able to enhance ODC levels in conjunction with cyclophosphamide, BBN and MNNG as initiators and with cyclophosphamide, saccharin and lithocholate as promoters. However, it did not enhance tissue ODC levels when administered alone. Bracken, shikimate and quercetin induced a macrocytic, normochromic, non-regenerative anaemia with thrombocytopenia and granulocytopenia at the termination of the 90-day study. The bracken-induced ataxia of monogastrics was attributed additionally to nitrite and cyanide, due to the very high levels found in the urine and serum respectively of bracken-fed rats over a 28-day test period. The high urinary nitrite levels of the bracken-fed rats prompted an investigation into the role of N-nitrosation in bracken-induced carcinogenesis. To this end, the Nitrosation Assay period (NAP) revealed the presence of nitrosatable entities in the gut of the bracken-, quercetin- and shikimate-fed rats and in the urine of the bracken- and quercetin-fed rats. This suggests that nitrosation, probably via the formation of nitrosamides, might be an important mechanism for the mediation of bracken-induced carcinogenesis. The plausibility of explaining various aspects of the sub-acute and chronic toxicity of bracken in monogastrics and ruminants on the basis of a "nitrosamide hypothesis" is discussed.

The effects of acute parenteral, repeated parenteral and dietary administration of cadmium chloride have been investigated in the male rat. A release of kidney enzymes into the urine occurred during the development of cadmium-induced renal damage, and provided a useful criterion of toxicity in further studies. Cadmium accumulation in liver and kidney was associated with increases in the concentrations of zinc and copper in these tissues. Dietary cadmium exposure depleted the renal and hepatic content of iron. Repeated parenteral administration of cadmium had a similar effect but only in the kidney, and produced an excessive loss of trace metals into the urine, which may have exacerbated the disturbances in their metabolism. Changes in the plasma concentration of trace metals depended upon the route of administration and dosage of cadmium. A single injection of cadmium chloride increased the concentration of copper in plasma but decreased that of zinc and iron, while repeated injections increased the concentrations of both zinc and copper, and decreased iron. Long term dietary administration of cadmium chloride raised plasma copper but decreased both zinc and iron. The administration of high doses of zinc sulphate altered the tissue disposition of cadmium. The possibility that zinc may prevent some of the toxic effects of cadmium was investigated, but zinc itself induced changes in the homeostasis of essential metals. Cadmium-treated rats showed changes in calcium homeostasis which were suggestive of an impaired absorption, and the accumulation of trace metals in the femur was inhibited. These changes were independent of the development of kidney damage. The dietary administration of cadmium also decreased the activity of femur alkaline phosphatase. This effect was prevented by dietary zinc supplementation. The effectiveness of a variety of chelating agents to mobilise and promote the excretion of cadmium was compared. Some lipophilic derivatives of diethylenetriaminepentaacetic acid were very effective when administered after acute cadmium exposure, but no compounds significantly decreased the body burden of cadmium in animals which had accumulated the metal in tissues.

The acute administration of AsIII or AsV to rats produced in liver a decrease in the heme saturation of tryptophan pyrrolase (TP) accompanied by dose-related increases in aminolevulinate synthetase (ALAS) and heme oxygenase (HO) activities and corresponding decreases in P-450 concentration. The relationship between heme synthesis and degradation was changed as a result of As treatment. The magnitude of these effects was related to the oxidation state of As, AsIII being more potent than AsV. These results support the contention that the heme saturation of TP is sensitive to treatments that modify liver heme concentration. However, it remains to be stablished which parameter, heme saturation of TP or ALA synthetase activity reflect more accurately changes in the "free heme" pool size. The increase in heme oxygenase produced by As in liver appears to be mediated by a mechanism largely or entirely independent of heme. There were indications that the inhibition of P-450- dependent monooxygenases reflects a selective effect of As on certain P-450 isoforms. Although only the higher doses of As were able to affect testicular heme metabolism, the general effect was similar to that observed in liver. AsV affected renal ALAS, HO and P-450 at doses which were ineffective in liver and testis. The main effects of continuous exposure to As III were : 1) an initial decrease in the degree of heme saturation of TP which remained constant during the period of treatment and 2) an initial increase in ALA synthetase which after 10 days of exposure was reduced to a 30% increase. No significant effects on heme oxygenase or P-450 concentration were observed. These observations were interpreted as indicative that a new balance between heme synthesis and degradation had been reached and that an adaptive response to the toxic effects of AsIII was taking place. Se produced effects on TP, ALAS and HO similar to those produced by As, but it was more potent than either AsIII or AsV. Se appears to be unusual in that it increases HO without reducing P-450 concentration. Se also appears to be the first element reported to increase epoxide hydrolase (EH) activity, as yet there is no evidence to explain how or why Se induces EH. It appears that Se has a dual effect on As-induced hepatoxicity. On the one hand it has additive effects in reducing the heme saturation of TP, and on the other it seems to protect against the decrease in P-450 levels produced by AsIII.

A keratinocyte culture derived from rat sublingual epithelium has been developed and characterised both morphologically and enzymically. Morphologically the culture closely resembles that found in vivo. The culture has been studied comprehensively using light and electron microscopy (scanning and transmission). The culture procedure has been optimised such that minimal amounts of time and materials are required. The culture, although heteroploid, shows a high degree of homogeneity with minimal fibroblastic contamination. Total adhered protein, total DNA content, 3H-thymidine incorporation, ornithine decarboxylase activity, acid phosphatase activity, prolinase and malate dehydrogenase activity have been studied at various phases in the cultures growth cycle. An initial study using 3,3',4,4' tetrachlorobiphenyl indicated that acid phosphatase, prolinase and total protein may be the best parameters for studying toxic insult in these cells, together with morphological examination by electron microscopy and light microscopy using acridine orange as a stain. Subsequently, comparisons of the 3,3',4,4' and 2,2,4,4' tetrachlorobiphenyl isomers, benzo(a)pyrene and 20-methylcholanthrene were made. Acid phosphatase and prolinase appeared to be useful markers of the toxic effects of these compounds showing alterations consistent with some in vivo findings. These changes were observed at concentrations of the compounds that did not produce significant cytotoxicity. Morphological examination showed changes with some similarities to some carcinomas in vivo. The effects of these compounds on keratin production within the cells has also been undertaken. A comparison has also been made of the relative toxicities of several compounds in the keratinocyte culture and in the human embryonic lung fibroblast line (BCL-Dl); the keratinocyte system appeared to be more sensitive to irritant compounds. Studies of the metabolic capabilities of the culture using benzo(a)pyrene as substrate have also been undertaken. Although the capacity of these cells to metabolise foreign compounds was low, they show hydroxylation, glucuronidation and sulphation activity. In longer term tests (14-21 day), the system shows enhanced sensitivity, the point of commitment of the culture (ie., differentiation and stratification) being a critical time. The methods used showed good reproducibility and were easily performed. The system may, therefore, provide a useful sensitive screening test for detection of compounds affecting differentiation (e. g irritants and carcinogens).

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