~ E ~ T RED R A (NR) ~ ASSAY
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Cl, 0.1 m1of 20 m M CaGl,, 0.3 m1of 50 mM sodium phosphate buffer 0.1 m1of 40 mM NADP (kept frozen until needed) and 0.2 m1of 10 rng mk" S9 protein. 3. Dilute S9 co-factor of mixture 1:lO into medium containing different concentrations of test agents; dilute controls 1:lO into medium without test agents. 4, Add 0.2-m1 aliquots of S9 co-factor/toxicant-amendedmedium to individual wells of a 96-well microtitre plate already seeded with cells and incubated for24 h. assay as described previously.
Medium without phenol red Standardized radiation source Lid of 96-well plate covered with black tape The NR assay can be used for studies on the effect of U7ir radiation on different cell types and the inhibition or enhancement of such effects by different xenoabich & ~orenfreund,1992).
1. Remove medium in which cells have been seeded to the 96-well plates, 2. Add medium without phenol red. This medium could contain various xenobiotics. 3. Expose to desired wavelength for p r e d e t e ~ i n e dtime intervals. Shield control 11s from radiation by covering wells with taped lid. place experimental medium with normal medium for desired time or again add a xenobiotic to be studied. Incubate at 37째C. 5. Add NR medium for 3 h for assay as described.
The NR assay was developed initially to serve as a potential alternative to the Drake rabbit ocular irritancy test (Borenfreund & Puerner, 1985). In addition to providing i n f o ~ a t i o non ocular irritancy, the assay was a good predictor of in vitro skin irritancy and acute toxicity. The NR assay has been used to evaluate the relative potencies of a spectrum of test agents, including inorganic metals, organometals, environmental pollutants, pharmaceuticals including cancer chemotherapeutics and over-the-counter drugs, surfactants, industrial chemicals, natural toxins, complex mixtures and biomaterials. Furthermore, this assay has been applied to investigations of metabolism-mediated cytotoxici~;structureactivity r e l a t ~ o n s ~ p s (SARs) for seriesof related chemicals; phototo~city;tempera t ~ r e - t o ~ ~ i t ~ i n t e ~ a ~ t i ~ n s ; s y n eand r ~ s tainc ~ a ~ o ~ ~ t i c i ~ t e r a between ctio~s s; nts; growth promo rs ands t ~ u l ~ t o ran ,1990,1991; B et al., 1993, 199