Autoradiographic Assay

In the first studies on the production of DNA repair in primary cultures of hepatocytes by chemicals, the protocol of applying the carcinogen first and then providing tritiated thymidine ([ H]-TdR) for incorporation during repair, as used by others,was employed. With this sequence, any repair performed before addition of [ H]-TdR would not be detected, and, indeed, simultaneous administration of carcinogen and [ H]-TdR resulted in greater repair than was produced by sequential application. 

This protocol is technically and logistically simple and has the important advantage that cells undergoing replicative DNA synthesis can be 

In evaluating a chemical, it should be tested over a dose range of 10 M (or the limit of solubility) to 10 M because repair is dose-related up to a point and then it falls off due to toxicity. 

TABLE 2. Background in Control Autoradiographs of Primary Cultures of Hepatocytes  

Background (grains/nucleus-sized area)  

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CD-I mice were exposed to purified air or benzene by inhalation at 0.04, 0.1, or 1.0 ppm for 22 hours per day, 7 days per week for 6 weeks (Ward et al. 1992). The effects of in vivo exposure to benzene were evaluated by using an autoradiographic assay to determine the frequency of mutants which represent mutations at the hypoxanthine-guanine phosphoribosyl transferase (hprt) locus in spleen lymphocytes. At the end of the six weeks exposure period, lymphocytes were recovered from the spleens of the mice and cryopreserved prior to assay. Mutant cells were selected on the basis of their ability to incorporate tritiated thymidine in the presence of 6-thioguanine. The increased frequencies of mutant spleen lymphocytes were significant at the low and mid, but not the high dose, and the method does not take into account possible clonal expansion. Further evaluation of the induction of gene mutation at these dose levels seems warranted. 

Greenamyre JT, Higgins DS, Young AB (1990) Sodium-dependent D-aspartate binding is not a measure of presynaptic neuronal uptake sites in an autoradiographic assay. Brain Res., 5II, 310-318. 

Citrullinemia is a human disease caused by a deficiency of liver arginino-succinate synthetase. It is inherited as an autosomal recessive. A fibroblast cell line from the skin of an infant with citrullinemia could not ultilize citrulline (instead of arginine) in the medium, while control normal fibroblast could (Tedesco and Mellman, 1967). For mutational studies, it may be possible either to use selective medium in which arginine is replaced with citrulline or to apply radioactive citrulline in an autoradiographic assay. 

The cells on the paper disc are viable and may be used in autoradiographic studies. Alternatively, they may be lysed by freeze-thawing and incubated in enzyme assays. 

Fatty acids released by lipases can be determined quantitatively by TLC, GC and HPLC. TLC methods are used in conjunction with densitometric methods or autoradiographic methods using radiolabelled TAG. Many GC and HPLC methods that have been outlined earlier are widely used to isolate and quantify FFAs in lipolytic assays. Additionally a method using p-nitrophenyllaurate as a substrate was described by Maurich et al. (1991) who quantified activity by the release of p-nitrophenol. Veeraragavan (1990) used a RP-HPLC method with triolein as the substrate. Triolein was emulsified in buffer with the aid of a surface active agent and the lipase added under controlled conditions. Lipolytic activity was measured by the release of oleic acid and quantified by absorbance at 208 nm. 

Radiolabeled bands on autoradiographs from Assays 1 and 3 can be quantified by densitometry and the activity of each sample graphed in arbitrary units. For Assays 2 and 4, counts in ADP-ribosylagmatine or nicotinamide are quantified directly and are converted to nanomoles of ADP-ribosylagmatine or nicotinamide formed/hour/mg protein, respectively, based on the specific activity of NAD in the assay. 

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