The objective of the proposed studies is to elucidate the physico-chemical characteristics and molecular mechanisms which determine the carcinogenic action of environmentally and occupationally important colloidal chromates. Using PbCrO4 as a prototype insoluble particulars chromate we will examine the relationship of particle size and shape to particle internalization using electron microscopy and cultured mammalian cells. The intracellular disposition of PbCrO4 particles will be examined by measuring dissolution products of 51Cr labeled particles in water, culture medium, microsomal preparations and intact cells. Intracellular distribution of 51Cr released from PbCrO4, will be measured by cellular fractionation and macromolecular binding studies. The relationship of particle internalization and dissolution to carcinogenicity will be examined in the Syrian hamster embryo cell colony assay (as a measure of early stage transformation) and the C3H/10T1/2 mouse embryo cell focus-forming assay as a measure of late- stage transformation. Cytotoxicity of PbCrO4 will be measured in colony- forming assays and by incorporation of radiolabeled precursors into DNA, RNA and protein. Toxicity to DNA function will be examined by measuring DNA replication and transcription of specific constitutively expressed and inducible genes. Damage to DNA structure will be assessed by alkaline elution and karyotypic analysis. The relationship of DNA damage to chromate mutagenesis will be examined using an integrated shuttle vector carrying a synthetic target sequence which can be rescued from host cell genomic DNA and analyzed by restriction and direct sequencing for frequency and type mutations. The target sequence will also be treated in vitro prior to integration (to form chromate-DNA complexes of DNA-protein crosslinks), lipofected into host cells, rescued after replication and analyzed for mutations. This allows for a direct analysis of the propensity of various types of chromate-induced DNA damage to result in base substitution mutations. The relationship of chromate mutagenesis to chromate carcinogenesis will be examined by using the polymerase chain reaction (PCR) technique to selectively amplify Ki-ras and Ha-ras oncogenes in chromate-transformed cells. The amplified fragments will be analyzed for mutations in the 12th, 13th and 61st codons by direct sequencing. These experiments will have practical value in evaluating the relative risk to humans in contact with specific colloidal chromates and will also help elucidate the molecular mechanism of chromate carcinogenesis and therefore carcinogenesis in general.
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