We have demonstrated that depletion or augmentation of GSH status to degrees matching or exceeding those measured in cardiovascular disease or cancer had no effect on the retention of
64Cu(ATSM) or its pharmacokinetic profile under either hypoxic or normoxic conditions. Furthermore, we have shown that inducing sufficient hypoxia to cause a significant increase in
64Cu trapping from
64Cu(ATSM) causes no measurable change in intracellular GSH status, at least in this acute experimental setting. Changes in intracellular GSH status within the limits of this study therefore do not appear to affect the hypoxia selectivity of
64Cu(ATSM), as had previously been suggested [
10]. In 1972, Petering reported that thiols directly reduce Cu-KTS; however, little has since been reported on the GSH-mediated reduction of Cu(ATSM) [
11]. GSH has more recently been demonstrated incapable of reducing Cu(ATSM) in an
in vitro system [
22]; however, this study did not preclude GSH acting as a cofactor in the (possibly enzymatic) reduction of the tracer inside the cell, nor did it replicate the relative concentrations of tracer and thiol that would occur
in vivo; to quantify Cu(ATSM) spectrophotometrically, it was necessary for Xiao et al. to use concentrations of Cu(ATSM) many orders of magnitude higher than we used in our experiments, which are closer to those occurring
in vivo during a PET scan. While BSO pre-treatment has previously been shown to not affect Cu(ATSM) retention in neuroblastoma cells under normoxic conditions, intracellular GSH concentrations in these experiments were not quantified, making it difficult to draw a definitive conclusion [
23]. In our study, therefore, we employed a sensitive radiometric technique allowing us to investigate Cu(ATSM) pharmacokinetics at tracer concentrations in a physiologically relevant model over which we have the capacity to accurately modulate (and confirm) intracellular GSH concentrations.
Intracellular GSH status changes dramatically during many disease processes in response to increased oxidative stress. In the heart, atrial glutathione levels have been demonstrated to be 58% lower in NYHA class IV patients than in healthy subjects [
14]. Experimentally, in isolated perfused rabbit hearts, the total GSH content fell by 54% after 90 min of ischaemia and by 61% 30 min after reperfusion. These degrees of GSH depletion are comparable to those we observe following BSO treatment in our model. The GSH content of cancer cells is, perhaps unsurprisingly, more variable. While adenocarcinomas and large cell carcinomas have been shown to have GSH levels approximately 27% lower than normal lung tissue, squamous cell carcinomas contain 207% of control values [
24]. The drug resistance of many tumours is thought to be mediated in part by GSH levels elevated up to 50-fold greater than normal [
25]–[
27]. With such large variations in intracellular GSH status in these disease processes, which
64Cu(ATSM) purportedly targets by virtue of their hypoxic status, it is essential to confirm that this hypoxia selectivity is not affected by variations in intracellular GSH content. While our results suggest that changes in GSH concentration do not contribute to the hypoxia selectivity of
64Cu(ATSM), we are not proposing that
64Cu(ATSM) reduction is not GSH-mediated. While it is still possible that other reductants may be responsible for this process (and that they may change during hypoxia to cause increased tracer retention), the intracellular concentration of GSH in mammalian cells is millimolar, and in vast excess to the sub-nanomolar intracellular concentration of
64Cu(ATSM) present when injected as a PET tracer. As such, even the significant augmentation and depletion of intracellular GSH content that we achieve in our experiments does not impact meaningfully upon this ratio. Intracellular thiols remain likely candidates fundamental to the intracellular reduction of
64Cu(ATSM), but their high relative concentration means that GSH-mediated
64Cu(ATSM) reduction is not a rate-limiting factor in radiotracer retention. It is currently unclear whether the rate of reduction or dissociation may change during hypoxia, perhaps influenced by changes in intracellular pH [
9],[
10],[
28], or whether the rate of tracer retention is purely governed by intracellular oxygen availability for reoxidation.