Handbook of Solvents - George Wypych - ChemTech - Ventech!

1096 James L. Botsford
In Europe many tests with animal and human cells have been developed (Clemendson
et al., 1996). These tests are effective, provide values comparable to those obtained with animals.
But they require that the animal cells be grown in culture. This requires a sophisticated
laboratory and well trained personnel. Again the tests take several days. Some tests
take up to a week. Damage to the cells is determined in a variety of ways. The ability of cells
to reduce the thiazole tetrazolium dye, MTT, is noted; the activity of an enzyme, often lactic
dehydrogenase, is determined; the ability of the cell to take up some dyes or to retain other
dyes is observed. Often morphological changes are noted to determine if the cells are damaged.
These techniques can be quite complicated and require well trained personnel and a
sophisticated, well equipped, laboratory.
There are several reports using animal cells to test the MEIC chemicals (Shrivastava et
al., 1992; Rommert et al., 1994; Rouget et al., 1993). Shrivastava et al., used freshly isolated
rat hepatocyte cells and two transformed cell lines. Comparable results were obtained with
the three cell types indicating that rat hepatocytes are no more resistant to toxic chemicals
than are other cells. They determined viability of the cells with 1) morphological studies; 2)
lactic dehydrogenase activity in the hepatocytes; 3) the ability of the cells to take up the dye
tryptan blue. They got much the same results with the three techniques. Rommert et al., simply
determined the effect of the toxins on the ability of the cells to replicate. They determined
cell numbers with a Coulter Counter after 48 hours. Rouget et al., followed the effect
of the toxins by determining the ability of cells to reduce the tetrazolium dye MTT and the
ability of cells to take up the dye neutral red. The authors maintained that the two methods
provided comparable results.
In Europe tests with rotifers, cysts from aquatic invertebrates (Calleja et al., 1994), development
of onion roots have been tried but none have been accepted (Persoone et al.,
1994; Snell and Persoone, 1989; Fresjog, 1985; Gaggi et al. 1995). Jaffe (1995) has proposed
a method using the survival of a protozoan. Toxic chemicals kill the protozoa. The
test requires a Coulter particle counter to determine the numbers of viable protozoa.
Tests using bacteria as the indicator organism are on the market. Microtox TM uses a
bioluminescent marine bacterium (Bullich et al., 1990). The bacteria, when growing in high
numbers, emit light. Toxic chemicals inhibit the production of light. The test requires a
luminometer to measure light production and a refrigerated water bath to grow the cells at
15 o C. The results are difficult to interpret. The ability of the cells to emit light gradually decreases
and a control to follow production of light in the absence of a toxin must be run. And
the value for this enters into the calculations. Typically the calculations require at least a
half hour to carry out (Ribo and Kaiser, 1987). A sophisticated computer program is used to
analyze the results. The initial cost of the kit is significant, but it is inexpensive to use once
this cost is absorbed (Elanabarwy et al., 1988). The test appears to be challenge for an MS
level graduate student at our university. It is not certain why toxic chemicals inhibit light
production but it is assumed that the chemicals damage the cytoplasmic membrane and reduce
electron transport to the pigments responsible for light production. This method is very
popular as a “first test”, to see if a chemical need be investigated further. The test has been
used to determine the toxicity of more than a thousand chemicals (Kaiser and Palabrica,
1991). There is a European version of the same text, Biotox TM (Kahru and Borchardt, 1994).
Thomulka et al. (1995) have proposed a variation on this test.
Polytox TM uses a consortium of bacteria from sewage sludge. Toxic chemicals inhibit
the oxidation of a carbon source (presumably glucose). Presumably the toxic chemicals
damage the cellular membrane and the associated cytochromes involved in electron transport.
Oxidation of the substrate is followed with a respirometer or with oxygen electrodes.
Both these methods are complicated and expensive to set up and to maintain. Again, at our
university, this technique appears to be appropriate for MS level graduate students. This test