Simultaneous photoreduction and Raman spectroscopy of red blood cells to investigate the effects of organophosphate exposure.

Simultaneous photoreduction and Raman spectroscopy with 532 nm laser has been used to study the effects of organophosphate (chlorpyrifos [CPF]) exposure on human red blood cells (RBCs). Since in RBCs, auto-oxidation causes oxidative stress, which, in turn, is balanced by the cellular detoxicants, any possible negative effect of CPF on this balance should results in an increased level of damaged (permanently oxygenated) hemoglobin. Therefore, when 532 nm laser, at a suitable power, was applied to photoreduce the cells, only common oxygenated form of hemoglobin got photoreduced leaving the permanently oxygenated hemoglobin detectable in the Raman spectra simultaneously excited by the same laser. Using the technique effects of CPF to build up oxidative stress on RBCs could be detected at concentrations as low as 10 ppb from a comparison of relative strengths of different Raman bands. Experiments performed using simultaneously exposing the cells, along with CPF, to H2 O2 (oxidative agent) and/or 3-Aminotriazole (inhibitor of anti-oxidant catalase), suggested role of CPF to suppress the cellular anti-oxidant mechanism. Since the high level of damaged hemoglobin produced by the action of CPF (at concentrations >100 ppm) is expected to cause membrane damage, atomic force microscopy (AFM) was used to identify such damages.Upper panel: Raman spectra of normal, photoreduced CPF exposed and unexposed RBCs. Lower panel: The weak Fe-O2 Raman band for CPF exposed cells shown on the left. The AFM images of unexposed and exposed cells are shown on the right. Scale bar, 2.5 μm.