This study sought to develop and validate a novel tissue culture medium-based biomarker of metabolically active live cells that could be used to quantify cell cytotoxicity to various agents and radiation. We had previously demonstrated using a state of the art HPLC electrochemical detector that live rodent cells convert HEDS into ME in vitro (Ayene et al., 2000
). Most importantly, we had demonstrated that the HPLC peak for ME was observed in the extracellular medium only after incubation of rodent and human cells with HEDS (Ayene et al., 2000
). In the absence of HEDS, the extracellular medium did not have any detectable amount of mercaptoethanol. These studies also demonstrated that the conversion of HEDS into ME is dependent on the activity of the oxidative pentose phosphate cycle (OPPC), suggesting that this assay could be used for all mammalian cells (Ayene et al., 2008
; Ayene et al., 2000
; Ayene et al., 2002
; Biaglow et al., 2003
; Biaglow et al., 2000
; Biaglow et al., 2006
). There are several biochemical pathways involved in the reduction of HEDS in live mammalian cells, presently below.
Direct reduction of HEDS by GSH and NADPH may produce ME, NADP and GS-adduct (reactions 1 and 2).
Normally, in cells, the generation of NADPH produced by oxidative pentose phosphate cycle (OPPC) recycles GSSG back to GSH (reactions 2 and 3), which is also involved in the reduction of HEDS (Biaglow et al., 2000
; Biaglow et al., 2006
We know from our studies and those of others that the reduction of HEDS is also facilitated by thiol transferase linked reactions (Bjornstedt et al., 1997
). However, the thioltransferase enzyme also utilizes GSH in the reduction of HEDS to stimulate the second step of reaction 1 (Bjornstedt et al., 1997
). The schematic representation in shows that five major metabolic pathways, which ultimately require OPPC for their activity, may be involved in “HEDS conversion into ME” ().
Biochemical pathways involved in biroreduction of HEDS
Based on the DTNB reaction of the metabolite of HEDS and our previous high pressure liquid chromatography and electrochemical detector (HPLC/EC) data that identified ME as the only DTNB reactive thiols produced by HEDS bioreduction (Ayene et al., 2000
), our current findings demonstrated that ME is the main product produced from HEDS in these human cancer cells (HCT116, HT29, MCF7, MCF10A, SKBR3) and CHO cells. Our results demonstrated that the metabolic conversion of HEDS into DTNB reactive product correlates with the cell number measured by Coulter counter. Additionally, the correlation between DTNB reactive product after HEDS incubation and cell number measured by Coulter counter showed a better linearity than currently available (XTT, WST-1, alamar blue, presto blue and cell titer glo) assays (). Although the results presented in – and , which were all carried out in 96 well formats, were sufficient to demonstrate the novelty and efficiency of the HEDS assay, we have also presented data for 6 well formats in , and . We have used six wells format to demonstrate that this assay could also be used as well as the Coulter counter in 6 wells format that is quite commonly used in determining the toxicity of radiation and chemotherapeutic agents.
Cisplatin is one of the most commonly used chemotherapeutic agents in humans (Barabas et al., 2008
) and there remains great interest in understanding the mechanisms of cancer cells resistance to platinum compounds. The normal tissue toxicity of platinum compounds is a dose limiting factor in cancer therapy (Barabas et al., 2008
). Addressing these issues requires a more efficient cell survival assay that could determine the changes in survival of cancer cells. We therefore tested the sensitivity of this assay in measuring the cell survival of human colon cancer cell HCT116 after cisplatin treatment. The results demonstrated that HEDS assay is suitable to determine the survival of cells after treatment with cisplatin in a 96 well plate.
Hydrogen peroxide (H2
) is used to understand the signaling pathways during oxidative stress and to screen/test the efficacy of antioxidants (Groeger et al., 2009
; Muller et al., 2007
). Because of the wide use of this oxidant, we determined the application of HEDS assay in quantifying the toxicity of hydrogen peroxide in human cells in a 96 well plate. The results demonstrated that HEDS assay can estimate the survival of cells after treatment with hydrogen peroxide. Although hydrogen peroxide is known to induce glutathione depletion, the HEDS assay worked as well as the Coulter counter suggesting that modest GSH depletion does not affect the HEDS assay. Consistent with this, our data with acetaminophen, which is also known to deplete GSH, demonstrated that acetaminophen induced toxicity measured by HEDS test is comparable to Coulter cell counter analysis suggesting that the GSH depletion, if any, is either not strong enough to affect the HEDS assay or GSH is regenerated by cells at the time of the assay. This is consistent with previous studies that have shown that the GSH depletion by acetaminophen is mild in HUH6 cells and regenerated to the control level after 5 hours in liver (Neuwelt et al., 2009
; Henderson et al., 2000
). However, strong GSH depletion persisted during the assay may have some influence on the measurement. Under these conditions, the HEDS test can still be equally effective if the time of incubation is increased longer than 2 hours or the assay is done after GSH regeneration.
The results with hydrogen peroxide and acetaminophen demonstrated that HEDS assay has no disadvantages in these cells under normal growth medium. However, it may be affected in zero glucose medium, G6PD deficient cells or cells completely depleted of GSH since glucose and G6PD and to some extent GSH are essential for the bioreduction of HEDS. However, this assay will be certainly useful for chemotherapeutic agents, oxidant, toxins and radiation or other cytotoxins that have mild effects on multiple pathways involved in the metabolism of HEDS. This is further confirmed by the current results that clearly demonstrated the application of HEDS assay for four different classes of cytotoxic agents (DNA damaging agents, oxidants, toxins, radiation).
Environmental pollution is a major concern worldwide. In the U.S., arsenical toxins rank third of all hazardous chemicals in highly polluted superfund sites. In ground water inorganic arsenic commonly exists as arsenate (As5+
) and arsenite (As3+
) and the reduction of As5+
may increase As3+
content in soil (Singh, 2006
). In air, the semimetallic form of arsenic oxidizes rapidly and at high temperatures will produce arsenic trioxide, a compound used in humans to treat certain types of cancer (Evens et al., 2004
). Phenyl arsenic compounds are the main contaminants in ground water at abandoned sites with a history of arsenic containing chemical warfare agents (Kroening et al., 2008
). The beneficial use of arsenic in cancer and potential harmful effects in humans at low exposure prompted us to explore HEDS assay for measuring arsenic-induced cell death. We therefore tested the application of the HEDS assay in quantifying the toxicity of PAO and arsenite in human cells (). The results demonstrated that HEDS assay can effectively measure the toxic effects of environmental pollutants in human cells.
Etoposide is a topoisomerase II inhibitor used in cancer therapy (Hande, 1998
). Etoposide is known to kill cancer cells by producing DNA double strand breaks (Hande, 1998
). Like many other chemotherapeutic agents, etoposide is also toxic to normal cells (Joel et al., 1996
; Kobayashi and Ratain, 1994
; Massimino et al.). The results demonstrated that HEDS assay is a good measure of the toxic effects of topoisomerase inhibitor in vitro.
The cellular response to radiation is also dependent on the oxygen concentration in the cells (Vaupel and Mayer, 2007
). Several previous studies have demonstrated that cells under hypoxic conditions are resistant to radiation as compared to that in aerobic condition (Vaupel and Mayer, 2007
). Hypoxic cells are quite common in most solid tumors, which determine the outcome of radiation therapy. Hypoxic cancer cells are also responsible for tumor vasculature and growth (Vaupel and Mayer, 2007
). Although certain drugs are used to target hypoxic cancer cells, there still remains a great need for a fast cytotoxic assay to measure hypoxic resistance that will be useful to screen better hypoxic drugs (Ahn and Brown, 2007
). The results demonstrated that HEDS assay can be used to measure the hypoxic resistance of cancer cells to radiation.
Our results suggested that this simple extracellular medium based biochemical assay could be used to determine the survival of cells that produce NADPH. The continuous recycling of NADP to NADPH, which requires a fully functional OPPC and glucose substrate, is essential to convert HEDS into ME. Glucose is converted into glucose-6-phosphate by hexokinase in all living cells. Glucose-6-phosphate is used as a substrate by G6PD/OPPC to produce NADPH. NADPH is used directly or as a cofactor to reduce HEDS into ME, which is released into the tissue culture medium. This conversion is dependent on the glucose level and active metabolic pathway of live cells. Unlike glutathione disulfide, HEDS is a unique non-toxic disulfide that is readily converted into a reduced thiol by glucose-dependent metabolic activity of live cells and transported into the extracellular medium in vitro. Our preliminary results demonstrated that ME produced from HEDS bioreduction is not toxic to cells under the conditions used in this protocol. Although ME is a well known reducing agents for proteins in vitro, it is less effective as compared to DTT in changing the function of protein in intact cells (Valetti and Sitia, 1994
). Further, it is unlikely to be toxic since it maintains the integrity of proteins and cells (Janjic and Wollheim, 1992
Our present results now demonstrate that this metabolic conversion of HEDS can be used to determine cell density and response of cancer cells to radiation, chemotherapeutics, chemical oxidant and environmental toxins. The HEDS conversion directly correlated to cell proliferation measured by Coulter counter suggesting that the conversion of HEDS into ME is a direct measurement of live cells. Dead cells will fail to convert HEDS into ME due to the loss of OPPC activity. This conclusion is further confirmed by our current findings that revealed a dose-dependent decrease in ME released into the tissue culture medium by cells treated with cisplatin, etoposide, hydrogen peroxide, arsenicals and radiation as compared to untreated cells. Our results demonstrated a direct quantification of cell death or loss of survival, since the loss of HEDS conversion correlated with decrease in cell number measured by the Coulter counter. Most importantly, the lack of conversion of HEDS into ME could be easily quantified by dithiobiznitrobenzoic acid, with the simple HEDS+DTNB biochemical assay taking less than 2.5 hours to complete in the laboratory.