Microregional heterogeneity of non-protein thiols in cervical carcinomas assessed by combined use of HPLC and fluorescence image analysis

Under low oxygen conditions, non-protein thiols (NPSHs, non-protein sulfhyd ryls) can effectively compete for DNA radicals sites and hence represent a potentially important cause of radiation resistance in the clinic, Intra- a nd intertumoral heterogeneity of glutathione (GSH) and cysteine were assess ed in cryostat sections of multiple biopsies obtained from 10 cervical carc inomas by the combined use of a sensitive high-performance liquid chromatog raphy (HPLC) method and a fluorescence image analysis technique to examine the spatial distribution of NPSHs in tumor tissue. Glutathione concentratio ns ranged from 1.98 to 4.42 mM; significant (greater than or equal to 1 mM) concentrations of cysteine, a more effective radioprotector than GSH, were found in some tumors. By HPLC, the intratumoral heterogeneity of NPSHs was relatively small compared with the intertumoral heterogeneity. The histoch emical stain 1-(4-chloromercuryphenoylazo)-2-napthol (mercury orange), whic h binds to GSH and cysteine, was used to determine the spatial distribution of NPSHs in tumor tissue. A comparison of NPSH levels in serial cryostat s ections showed a close correlation between NPSH values determined by HPLC a nd mercury orange fluorescence quantification. Using fluorescence image ana lysis, an similar to 2-fold increase of NPSHs in tumor versus nonmalignant tissue was observed in the same section. Because some cervical carcinomas c ontain radiobiologically important levels of cysteine, agents that target t he biochemical pathways maintaining tumor cysteine have therapeutic potenti al as adjuncts to radiotherapy in cervix cancer patients.