Handbook of Solvents - George Wypych - ChemTech - Ventech!

1110 James L. Botsford
Table 15.2.2.6. Effect of high concentrations of EDTR and calcium on toxicity
(Inhibition in %)
Compound
2.5 μmole EDTA, 0.74 μmole Calcium
Control + EDTA, + Ca
25 μmole EDTA, 7.4 μmole Calcium
Control + EDTA, + Ca
isonicotinic acid 27.0 25.4 30.9 105
2,4-dinitrophenol 44.7 63.0 58.2 50.0
Streptomycin 55.0 48.9 47.2 0.00
Neomycin 0.037 62.4 77.2 77.3
pentachlorophenol 48.0 63.7 26.0 13.3
3-phenoxybenzoate 54.3 74.0 34.5 37.8
15.2.2.9 Mechanism for reduction of the dye
It is not known how the dye is reduced. It is not known why toxic chemicals inhibit the reduction.
It is thought that tetrazolium dyes are reduced by cytochromes (Altman, 1975). But
this has been questioned (Marshall et al., 1993). In eucaryotic cells, all the cytochromes are
in the mitochondria. Marshall’s group has found that the dyes are reduced in preparations
from cells with the mitochondria removed. It has been found that a mutant of Escherichia
coli lacking one of two major cytochromes found in this bacterium is unable to reduce MTT
(Botsford, unpublished). But in E. coli, reduction of the dye is not inhibited by toxic chemicals.
This may not be an analogous situation. The dye could be reduced by a different mechanism.
R. meliloti, like most other bacteria, has many reductases. Some of these are membrane
associated and damage to the membrane could affect the reductase. One of these reductases
could be responsible for reduction of the dye. It has been found that the MTT is
transported into the cell before it is reduced. The reduced dye is inside the cells. Cells with
the dye can be concentrated by centrifugation, the dye appears in the cell pellet. None of the
dye is in the supernatant. Toxic chemicals could interfere with the transport of dye into the
cell prior to reduction.
Transposon insertion mutants unable to reduce the dye have been obtained and five
mutants have been isolated. All grow very slowly in minimal media supplement with 0.1 %
casamino acids and obviously all have lost a critical function. With these mutants it should
be possible to clone and then to sequence the function responsible for reduction of the dye.
From the sequence, the nature of the function can be determined.
In our studies comparing the Rhizobium assay with other assays, it was observed that
tests using viable animals were almost always less sensitive to toxins. Tests using Daphnia,
the various animal cell tests and Microtox TM and the Rhizobium test seemed to be most sensitive.
Tests using fish (fathead minnow, trout fingerlings) give results comparable to tests
with the bacterial indicators. Polytox TM and QSAR were less sensitive, had higher IC50 values,
but were more sensitive than the tests using viable animals. Were the author asked to
recommend a test procedure to indicate if a chemical were toxic, the author would recommend
an animal cell test, probably using freshly isolated rat liver hepatocytes, the
Rhizobium test and the Microtox TM test. Tests with freshly isolated rat hepatocytes would
not require that the cells be grown in a laboratory situation and this would be much simpler.
These three procedures are much more sensitive than tests involved live animals. These
three tests would be simpler than tests with Daphnia. This would provide a “battery of