An approach to the validation of biomarkers of harm for use in a tobacco context.

There is both a call and a need for biomarkers of harm that are validated for use in a tobacco context. Currently, there are no validated biomarkers and there is no consensus about which ones may be suitable for this purpose. To advance the science in this area a working definition of biomarkers of harm and a shortlist of candidate biomarkers are proposed. A framework for the validation of biomarkers of harm using of a series of epidemiological studies culminating in a targeted prospective study is outlined. The candidate biomarkers have advanced to preliminary testing although this does not imply that any on the shortlist will become validated. This framework could also be used for the evaluation of proteomic, genomic, transcriptosomic or metabonomic profiles, which may turn out to be the preferred biomarkers for use in harm prediction. Biomarker studies would complement data that are generated from specific in vitro tests and from animal studies to evaluate tobacco products.

Zonal changes in the rat liver following an acute dose of phenobarbitone: an ultrastructural, morphometric and biochemical correlation.

Alterations in the liver of rats 6 h after a dose of phenobarbitone have been studied by subcellular fractionation, conventional electron microscopy and morphometric analysis. The area immediately surrounding the central vein was the only area to undergo any alterations. There was a morphometrically measurable but not observable cellular hypertrophy of 71% whilst the hepatocyte complement of rough endoplasmic reticulum (RER) and smooth endoplasmic reticulum (SER) was increased by 72% and 93% respectively. The increases in RER and SER were not apparent by observation and it is assumed that they have been diluted by the cell hypertrophy to 1% and 22% which must be below the threshold for detection by subjective observation. Following subcellular fractionation and measurement of microsomal protein, there was no significant difference in the level of microsomes isolated from control or treated rats. Therefore, the morphometrically measured increase in RER and SER would appear to be restricted to a relatively small population of hepatocytes adjacent to the central vein. Such an increase would represent only a small percentage of total microsomes in a homogenate and would almost certainly be masked by variation in animals and techniques. Disruption of RER was also observed in hepatocytes that would proliferate their SER should phenobarbitone treatment have been continued. Therefore this RER disruption would seem in no way to interfere with the process of membrane and enzyme synthesis.

Zonal changes in the rat liver after chronic administration of phenobarbitone in ultrastructural, morphometric and biochemical correlation.

Alterations in the liver of rats subjected to 24 days of continuous administration of phenobarbitone have been supplied bu subcellular fractionation, conventional electron microscopy and morphometric analysis. The increase in wet weight of the liver was found to result from a combination of cellular hypertrophy, hyperplasia and an enlarged hepatic blood space. In the centrilobular zone all the hepatocytes underwent a substantial proliferation of total ER, became enlarged and had an increased blood supply. However, in the periportal zone phenobarbitone caused changes in only 45% of the hepatocytes, the remainder being apparently resistent or tardy. An overall dramatic increase in hepatic RER was both measured and observed but the response involved hepatocytes in which the RER had proliferated as well as those which were depleted of RER or had stacks and cisternae that were severely shortened and dispersed. These alterations are discussed in relation to changes in RER after administration of agents causing hepatonecrosis. Possible reasons for the inability of other workers to detect a phenobarbitone-induced increase in RER are also put forward. After subcellular fractionation and corection for centrifugation losses into the 9500 g pellet, using the microsomal marker cytochrome P-450, phenobarbitone-induced increase in total ER was substantially less than that found by morphometric analysis. This indicates that during the preparation of microsomes a substantial proportion of intracellular membranes, having different metabolic and synthetic properties to those finally isolated, are discarded and emphasizes the need to exercise care when using microsomal preparations.

Microagglutination test for detecting and measuring serum agglutinins of Francisella tularensis.

A microagglutination method utilizing stained antigen for detecting and measuring serum agglutinins against Francisella tularensis is described. The microagglutination and standard tube agglutination techniques were demonstrated to be comparable in sensitivity and specificity. Advantages of the micro method are rapidity and ease of performance, economy of reagents and, in particular, ease of interpreting specific reactivity.