We used the
oxygen sensitivity of the histochemical reaction to detect
glucose-6-phosphate dehydrogenase (G6PDH) activity based on
neotetrazolium (NT) reduction to discriminate
cancer cells from normal cells.
Formazan generation was strongly reduced in normal but not in malignant cells when the incubation was performed in
oxygen instead of
nitrogen. Competition for reductive equivalents between NT and
oxygen via
superoxide dismutase (SOD) has been suggested. Since SOD activity is usually decreased in
cancer cells, NT reduction would not be hampered in these cells. We tested this hypothesis by demonstrating
NAD-dependent
lactate dehydrogenase (LDH) activity instead of
NADP-dependent G6PDH activity in normal rat liver and colon, in human colon
carcinoma, and in experimentally induced
metastases of colon
carcinoma in rat livers. Reactions for both
enzymes were determined cytophotometrically in an atmosphere of pure
oxygen or
nitrogen. G6PDH acted as described previously, showing distinct activity in
cancer cells but strongly reduced activity in normal cells after incubation in
oxygen, but this was not the case with LDH because
formazan was also generated in normal tissue in
oxygen. It appeared that after 5 min of incubation at 37 degrees C the residual activity of G6PDH in an atmosphere of
oxygen compared with
nitrogen was 0% in normal liver tissue and 15% in normal colon epithelium, whereas in colon
carcinoma and in colon
carcinoma metastasis in liver it was 48% and 33%, respectively. The residual activity of LDH in
oxygen was 30% in normal female rat liver, 75% in normal male rat liver, and 38% in normal colon epithelium, whereas the residual activity in colon
carcinoma and
metastases in liver was 54% and 24%, respectively. These experiments clearly indicate that the
oxygen sensitivity phenomenon is not solely an effect of competition for reducing equivalents between NT and
oxygen via SOD, because
NADPH generated by G6PDH and
NADH generated by LDH have a similar redox potential. Apparently the system is more complex. The role of specifically
NADPH-converting cellular systems such as
NADPH-cytochrome P450 reductase was excluded because incubations in the presence of exogenous
NADPH as substrate for these systems revealed
oxygen sensitivity. Involvement of
NADPH-dependent lipid peroxidation in the
oxygen sensitivity test is discussed.