Abstract
In this investigation in vitro systems were employed to study the biological effects of transfer, metabolism, cytotoxicity and mutagenicity of one PAH, benzo(a)pyrene a known mutagen and carcinogen, in different physical states in association with various particles. The physical forms in which BP was studied were, particulate-adsorbed, microcrystalline and in a dissolved state (in an organic solvent dimethylsulphoxide (DMSO) and aqueous buffer (PBSA)). The particles onto which BP was adsorbed were alumina, TiO2 and Fe2O3. The Ames (bacterial mutagenicity) test gave a direct measure of the potential biological response of the particles themselves and associated with BP in its adsorbed and non-adsorbed states. Particulate adsorbed BP produced enhanced mutagenicity compared to BP alone, when dosed in PBSA. Alumina B (the high surface area alumina) produced the greatest mutagenicity. The mutagenic effects of BP in the Ames test required metabolism to epoxides, diols, tetrols and quinones (See Section 1. 8). The extent of this response was reflected by the availability of BP from its various states to the biological systems (microsomes). The physical state on the surface of the particle and the availability of the BP to the microsomal membrane bound enzymes was indicated by measuring the transfer of BP into the microsomes, by the method described by Lakowicz et al (1979), which involved the fluorometric measurements of benzo(a)pyrenes. BP was found to be in both monomeric and exitomer (crystalline) states on the surface of the particles. The alumina B showed a prominent presence of monomeric BP compared to other particles and the greatest transfer rate. The metabolic profile investigation was carried out using High Performance Liquid Chromatography (HPLC) and 14C-radiolabelled BP. Examination of BP’s metabolite profile reflected the effects of the particles on the various states of BP and its availability. Indication of favoured metabolism and inhibition of part of the cytochrome P-450 enzyme system was not seen. The extent of metabolism was indicative of the BP available, which was related to the BP transfer rates. BP coated alumina B was the most extensively metabolised of the BP coated particles. In addition to these subcellular studies, the effects of the particles and BP adsorbed particles were investigated using an established macrophage-like mammalian cell line P388D1 (Dawe & Potter, 1957; Koren, et al 1975), and which has been used as a favourable model of cytotoxicity index, of macrophage function (Daniel et al 1980; Wright et al 1980 and Wade et al 1980) and hence that of possible lung toxicity. A comparison with mice peritoneal macrophages was also made. A parallel assessment of cytotoxicity in a non-phagocytic cell line (CHO and V79) was also performed. The indices of cytotoxicity used were release of Lactate dehydrogenase (LDH) and lysosomal enzymes (B-Gal, B-Gluc) and percentage of cell survival using Neutral Red and Kenacid Blue; as a result of exposure to particulate and BP associated particles. The limited cytotoxicity was observed with BP coated and uncoated particulates. The addition of a metabolising system (S9) produced cytotoxicity, although this was only a tentative observation as limited testing was performed. To compliment the in vitro work, an in vivo study based on the intratracheal instillation work of Saffiotti et al (1968,1972) and Henry et al (1973) was performed. This involved a single intratracheally instilled dose of ferric oxide particles coated with BP (in a 1:1 proportion) into male Syrian golden hamsters. Through a modification of the organ culture technique of Placke et al (1987), a tritiated thymidine incorporation over a 12hr to 5 day time period, was performed. Although no unscheduled DNA synthesis was detected, large numbers of S-phase cells were seen; the majority being in the trachea, and the BP-Fe2O3 dose having the greatest effect.