Zerler, B., E. Roemer, H. Raabe, A. Reeves and J. Harbell. Evaluation of the Phototoxic Potential of Chemically Modified Tetracyclines Using the 3T3 Neutral Red Assay. ATLA 1999. 27: 107.

Chemically modified tetracyclines (CMT), which lack antimicrobial activity, have the ability to inhibit matrix metalloproteases and, in some cases, serine proteases, thus attenuating connective tissue degeneration. Several of these compounds are now under development. Since tetracyclines are known to be photoactive (and some phototoxic), an in vitro screening programme was begun. To identify potentially phototoxic CMTs, the 3T3 neutral red phototoxicity assay was employed (Spielmann et al. Toxicology in Vitro 12, 305-327, 1998). 3T3 cells were seeded onto 96-well plates and incubated overnight. The growth medium was removed and replaced with phenol red-free Hanks’ balanced salt solution containing serial dilutions of the CMTs (two plates per compound). After initial incubation for 1 hour at 37 C, one plate was exposed to 5 J/m2 UVA/white light from a solar simulator, while the other was kept in the dark. The plates were then rinsed, re-fed, and incubated for 24 hours. Cell viability was measured by neutral red uptake. Phototoxicity was measured by the relative toxicity between the doses with, and without, light exposure following published guidelines. Reference compounds included commercially available tetracycline, doxycycline, and minocycline. The phototoxic response of the compounds in the present assay was consistent with their behaviour in vivo. These phototoxicity results, combined with efficacy data, facilitate rational choices in selecting compounds for further study development.