Method for rapid screening of teratogenic agents

The invention relates to a process for screening a compound for teratogenic activity which comprises administering the compound to a test animal and using amniotic fluid from the test animal in a cell culture medium. The compound tested is screened on the basis of cell growth in the culture medium.

The present invention relates to a novel method of screening substances 
including drugs, environmental pollutants and other substances with 
potential effects on biological systems for their possible deleterious 
effects on the development and normality of the mammalian foetus including 
the human foetus after exposure of the maternal organism to the substance 
in question. 
BACKGROUND 
Current tests of new drugs and other agencies for possible teratogenic 
effects on the human foetus are slow and expensive. Such tests are 
generally performed by the examination of some thousand rodent foetuses of 
various ages from pregnant females injected or fed with suspected 
teratogenic substances at different stages of pregnancy and at different 
dose levels. Other mammals are sometimes used. The embryos are examined 
for morphopathology and for hisopathology. Different periods of foetal 
sensitivity and different levels of susceptibility between strains and 
species characterise the response to teratogenic agencies. For this reason 
some investigations have used more than one species. Nevertheless the 
applicability of tests on rodents etc. to effects expected in man must 
always remain in doubt, since there are differences in placental structure 
and physiology as well as pharmacogenetic differences between species, yet 
tests on primates are even more expensive and time consuming than rodent 
tests, and are therefore not often undertaken. 
The costs to industry (excluding all overheads) even in 1975 were of the 
order of several thousands of pounds per substance tested in such a way. 
Each substance requires about a year of laboratory time. It is 
understandable therefore that information regarding teratogenicity 
sometimes remains to be deduced from epidemiological studies of human 
populations, after some family tragedies have already occurred. 
Since the number of potentially teratogenic substances increases faster 
than the capacity for testing by methods using intact animals, alternative 
forms of screening have been considered by several authors including organ 
or cell culture systems in vitro. However, three fundamental problems 
remain. 
A very serious problem is the form in which the substance should be 
presented to the cultures, since some substances administered to the 
mother in vivo do not exert their teratogenic effect directly on the 
foetus, but through a toxic or persistent metabolite: and may be harmless 
in vitro. Conversely, a substance toxic in vitro may be metabolished 
rapidly to a harmless agent in vivo. Furthermore, relevant dose levels for 
a particular group of susceptible cells may not be related in any obvious 
way to the dose administered in vivo. 
The method of scoring of effect presents a problem, since organ cultures 
require histological investigations and growth measurements which are very 
time-consuming, while cell cultures are difficult to score objectively, 
except for cytotoxic effects which are frequently associated with 
non-physiological levels of substances in the medium. Therefore, the 
method of scoring of results must exclude cytotoxicity if it is to be 
discriminating or morphology of the cells if it is to be objective 
A suitably responsive test cell system is required. 
The method 
A procedure is described below which avoids these problems. It is also 
inexpensive (less than .function.100 per substance tested at 1975 prices) 
and is rapid, objective, discriminant and data obtained from the use of 
non-primate material is capable of being confirmed by using primate (e.g. 
human) cells where indicated. It may also, in principle, be applicable to 
very small amounts of human foetal tissue (by-passing, if necessary, tests 
on non-primate systems). 
According to my invention I provide a process for screening a compound for 
teratogenic activity comprising administering the compound being screened 
to a test animal, removing amniotic fluid from the test animal, using the 
amniotic fluid removed as part or all of a cell culture medium, and 
measuring the effect on cell properties of cells grown in the culture 
medium containing the amniotic fluid from the test animal as compared with 
the use of amniotic fluid from an otherwise identical control animal that 
has not had the compound being screened administered to it. 
In more detail, in its generally preferred aspect, the procedure I have 
devised and tested is as follows: 
(1) Amniotic fluid is used as cell culture medium or as an additive to a 
standardised cell culture medium in order to test the substance as 
processed by the maternal organism. Amniotic fluids either from animals of 
diverse genotype or of selected genotypes, as required, are pooled. 
Controls are provided by amniotic fluids from uninjected/ 
untreated/unexposed individuals and the assays are provided by amniotic 
fluids from individuals exposed, injected or otherwise treated withbthe 
substances to be tested. Amniotic fluid is harvested shortly after 
exposure to the agent (e.g. 1-2 hours after administration or according to 
the rate of uptake). 
The substance tested is thus presented to the cells in the form and at the 
dose to which the foetus would be exposed, and problems due to different 
periods of feotal sensitivity are also bypassed. 
(2) A standardised cell suspension is innoculated into cultures and grown 
during log phase in the medium containing amniotic fluid from a control or 
test series as described above. Foetal cells are normally used but cells 
from embryonic or other stages may be used instead where appropriate. 
Cells in lag or confluent phase may be exposed where appropriate. Cells 
are labelled for a short period (not more than a few hours) before being 
harvested, with C.sup. 14 amino acids. A cell system is required which is 
sensitive to changes in metabolism while remaining viable. Lens epithelium 
has been used since I have published evidence that quantitative regulation 
of specific syntheses is of especial importance in this orgen and the 
assay system described in (3) below detects changes in mitotic rate, cell 
mass, and growth conditions, cell surface modification and genetically 
determined pathology. 
However, since it is possible that some substances may be found to which 
this tissue system is not sensitive, other cell culture systems such as 
fibroblasts, neural cells, endothelial or epithelial cells, etc. may be 
used instead of lens epithelium and are preferably used in addition to it 
for a fuller battery of tests. 
(3) An assay method which is rapid, repeatable, objective and quantitative 
is provided by the measurement of the profiles of accumulation and 
synthesis of individual proteins of the test cells (the synthesis being 
measured after incorporation of radioactive precursors before harvesting), 
as demonstrated by the densitometric quantitation (using any suitable 
accurate commercial densitometer) of the cell contents after resolution by 
high-resolution separation, e.g. by isoelectric focussing in 
polyacrylamide gels or other stabilising media in dissociating conditions 
(e.g. by using 6-8M urea) and staining with any of the quantitative 
protein stains (or in principle by absorbance at appropriate wavelength 
with unstained material). Similar densitometry is made of the 
autoradiograph of the gels. Each analysis can be done on a culture grown 
from a cell suspension of as little as 1-4.5 .times. 10.sup.5 cells, but 
can be scaled up or down as required. 
Cell contents for analysis may be extracted by any standard procedure such 
as freezing and thawing, sonication, homogenisation etc. 
This procedure has been found, in tests on two teratogens in the mouse (6 
aminonicotinamide and salicylate), to distinguish reliably and 
specifically between the cells treated by each of these teratogens, and 
control cells. It has also been used successfully on 
methadone-hydrochloride, methanol, corticesteroids and antihistamines in a 
pilot study. The method can be adopted for use in a primate system for 
further testing of any substances giving cause for concern (primate 
includes human). 
The test has also correctly detected the teratogenic compound from two 
coded compounds of unknown nature which were supplied for test by a 
pharmaceutical investigator from a major drug company. Using a panel of 
four foetal cell types the nature of the defects of exposed foetuses was 
also correctly deduced. The non-teratogenic compound was the one found 
harmless by this test. 
Time usually required for one assay, 11-15 days. However, by using 
fluorography, according to Bonner and Lasky, 1974, (European Journal of 
Biochemistry, 46, 83-88) instead of autoradiography the test may be 
completed in 9-10 days.

The following Example illustrates the invention. 
EXAMPLE 
Amniotic fluid 
Mice of numerous genotypes were taken from amongst all the more prolific 
strains available from the Institute of Animal Genetics Mouse House (Mouse 
News Letter, 1974). They were mated and the pregnancies dated by vaginal 
plug. They were injected once, subcutaneously, with a teratogen, between 
12-15 days of gestation in order to obtain a large volume of clear 
amniotic fluid. 1-2 hours after the injection the mice were killed by 
cervical dislocation, the uteri removed and the amniotic fluid harvested 
under sterile conditions, passed through a Millex filter and stored at 
-20.degree.C. Control amniotic fluids were obtained from uninjected mice 
of a similar range of genotypes and stages of gestation. Mice were 
injected with double the teratogenic doses used for 6-aminonicotinamide by 
Pinsky and Fraser (Biol. Neonat., 1,106-112 (1959)) and for sodium acetyl 
salicylate by Larsson and Erickson (Acta. Paed. Scand., 55, 569-576, 
)1976)), but the amniotic fluids were diluted to 50% with culture medium 
for use, thus giving a final concentration similar to that used by these 
authors. Five batches of pooled amniotic fluid of each kind were prepared 
in this way and tested in separate culture experiments. 
Tissue culture 
Cultures of dissociated lens epithelial cells were prepared according to 
Okada, Eguchi and Takeichi (Dev. Growth Diffn., 13,323-326 (197l)), and 
plated at 4.5 .times. 10.sup.5 cells per dish in 3.5 ml Earles MEM 
(Biocult) containing 6% foetal calf serum (Gibco). The medium was changed 
at 2 days, and after 4 days, when these cells were in log phase, the 
medium was replaced by 1 ml of medium and 1 ml of amniotic fluid. After 
two further days the cells were labelled for 4 to 5 hours with .sup.14 C 
mixed amino acids (Amersham) at 20 .mu.Ci/ml. The cultures were washed 
several times in Hanks solution, harvested with a miniature plastic 
scraper and taken up into a drop of 8M urea containing 10 mM 
.beta.-mercaptoethanol. The drop was frozen and thawed three times to 
liberate the cell contents. When required, the living cells were examined 
with a Gilbert and Siebert inverted phase microscope. 
Analysis of Protein Content 
Polyacrylamide gels containing ampholines according to Wrigley (Science 
Tools, 15, 17-23, (1968)) were modified to contain 6M urea, 10mM 
.beta.-mercaptoethanol, and an ampholine mixture giving a gradient from pH 
3 to 10, flattened in the region of 4.5 to 7.0. Such gels give especially 
high resolution for crystallins and are now routinely used in my 
laboratory. 20 .mu.l of cell preparation was added to each gel which was 
electrofocussed overnight according to Wrigley (loc. cit.). Gels were 
stained in Coomassie Blue, scanned with a Joyce-Loebl densitometer, sliced 
longitudinally, dried down onto Whatman No. 3 paper, in a modification of 
the method of Daniel and Wild (Anal. Biochem, 35, 544-545, (1970)) and 
clamped to a sheet of X-ray film. After 4 to 5 days of exposure the 
autoradiograph was developed and scanned. The entire procedure, from cell 
culture to final scan, was completed in 11-12 days. 
Results 
The protein profile of the lens epithelial cells grown in medium containing 
amniotic fluid is the same as that in medium without amniotic fluid. Cells 
grown in amniotic fluid from mice injected with 6-aminonicotinamide had a 
modified cell outline and a consistent set of changes in the protein 
profile, one band being missing and one exaggerated; while some other, 
less striking quantitative changes were also recorded. A few were probably 
no greater than random fluctuations, but many were seen regularly. Cells 
grown in amniotic fluid from mice injected with sodium acetyl salicylate 
are normal in morphology (unless they and their controls are grown in 
sub-optimal conditions). In all cases they were lacking in the band which 
was enhanced by 6-aminonicotinamide treatment. Other quantitative changes 
were also observed. Quantitative changes in the autoradiograph traces were 
found which distinguish equally well between the effects of these two 
drugs. Changes in the stained gels indicate changes in accumulated 
protein, those in the autoradiographs, changes in protein synthesis. These 
changes are presumably related to each other.