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The author describes an in vitro teratogen assay that uses Drosophila embryo cell cultures. The endpoints selected for assessing the teratogenic potential of any agent, physical or chemical, involve detecting inhibition of normal muscle and neuron differentiation and induction of heat shock (stress) proteins. Results so far suggest the Drosophila assay is capable of accurately establishing if a particular agent tested can act as teratogen by a variety of appropriate endpoints. Furthermore, this assay can be used not only as a screen to identify teratogens but also in mechanistic studies of abnormal development, gene involvement in resistance to teratogens and the possible role of heat shock (stress) proteins in preventing or minimizing birth defects.
Teratogens are estimated to account for four to five percent of all human birth defects. However, the actual percentage may be higher if one considers that many subtle mental deficiencies go undetected at birth and in early childhood.
The tragic consequences of using inadequately tested drugs or ones where there exists species-response variability was seen in the thalidomide babies born in the early 1960s. Current drug-testing procedures, mostly animal reproduction (in vivo) studies, are time consuming, expensive, and yield responses that may vary from species to species. With 50,000 to 70,000 different chemicals already in the market place and some 200 to 400 new drugs being produced every year, alternatives to whole animal, or in vivo studies are needed.
Two groups have provided the largest impetus for developing alternative testing methods (assays): the chemical manufacturing industry, which is interested in efficiently testing their products, and the alternatives to animal testing groups, who are very committed to reducing animal use in routine types of testing.
Numerous reviews have been published, surveying and or assessing the merits and shortcomings of the large number of in vitro assays that have already been developed, mostly during the last fifteen years (Faustman, 1988; Brown and Freeman, 1984; Brown, 1987). The majority of these in vitro models have the following elements in common: they are based on the consensus that teratogens can have a variety of effects on the developing embryo and at various organizational levels such as cell morphogenesis, mitosis, determination, cell-cell recognition, migration, cell death, nucleic acid synthesis, and protein synthesis. The final result of this disrupted development may range from death of the organism to birth defects. Although it is both unlikely and unnecessary that any one single in vitro system be sufficient, it is hoped that the successful development and validation of several such assays would greatly benefit many areas of teratology, especially as prescreens and possibly provide for mechanistic studies.
A chemical is classified as eliciting a teratogenic response if it results in a statistically significant reduction in the number of myotubes and ganglia when compared with controls. A 50% reduction in the number of myotubes and/or ganglia is taken as a positive response. The same chemical is tested in three or more separate trials before being classified as a teratogen or non-teratogen in the Drosophila culture system. Over 150 compounds have been tested in this assay, with a relatively small number of false positives and false negatives. Table 1 summarizes the results for a small but representative sample of the chemicals tested and compares them with published human and animal data.
A strong correlation exists between drugs that inhibit embryonic differentiation in vitro and those that induce the synthesis of two small heat shock proteins (Table 2). It is quite interesting that treatments, such as ether, heat shock, or exposure to some of the metal ions, induce the full complement of the heat shock proteins. These results indicate the induction of the heat shock proteins is under variable regulatory control.
A recent extension of assessing hsp 22 and 23 induction, and certainly a simplification of this molecular-level assay, has been to utilize what are termed "reporter-gene" constructs. A transgenic fly stock, in which a recombinant DNA has been inserted that contains DNA from another species, a rather useful molecular splicing event, is established that contains a reporter gene. In this case the hsp 23 gene promoter is linked to bacterial galactosidase gene. This construct comes under eukaryotic gene transcriptional control, that is, whatever molecular signal turns on hsp 23 transcription will now also turn on galactosidase production. This assay has been used extensively in developmental and genetic studies in Drosophila and more recently with transgenic mice. The levels of galactosidase are assessed by using a chromogenenic substrate that when cleaved results in a blue precipitate. Thus, cells that are exposed to a teratogen would turn blue. This is a very fast and efficient means to test the teratogenic potential of both physical and chemical agents. All chemicals previously shown to turn on hsp 22 and hsp 23 transcription have also been shown to turn on this reporter gene (Bournias-Vardiabasis, 1994, submitted for publication). This type of assay, commonly referred to as X-gal, could easily be adapted to be utilized as a Tier 1 teratogen screen to deal with the large number of untested chemical and environmental pollutants. Preliminary trials have indicated that we can also test for the presence of teratogens in polluted water sites (unpublished observations).
Again, Drosophila has the distinct advantage of having an extensively studied, well characterized genetic system. Several neurotransmitters have been identified in the nervous system of Drosophila. Acetylcholine has received the most attention, since it appears to be the major neurotransmitter system in the central nervous system in Drosophila (Salvaterra, 1988).
There has been little systematic study of nerve-specific or
muscle-specific
teratogens. Usually one finds isolated reports without extensive follow-ups.
Several years ago, McBride et al. (1983) demonstrated that pregnant marmoset
who had ingested tha-lidomide produced fetuses with reduced size and number of
neurons. In adults, thalidomide is believed to produce peripheral
neuropathy by interference with cholinergic nerves and will produce
malformations. Certainly the increasing evidence points to a role
neurotransmitters may play in regulating cell movement during tissue
morphogenesis (Zimmerman, 1985).
We have previously reported on the in vitro neuronal differentiation of Drosophila embryonic cells on expression of acetylcholine enzymes choline acetyltransferase (ChaT) and acetylcholinesterase (AchE) and on the effect that some anti-cholinergic agents have on normal patterns of neurotransmitter expression (Salvaterra et al., 1987). Those studies have been extended to test a number of neuroteratogens and assessing not only their morphological effects, but also their effect on ChaT and AchE expression. Table 3 represents a partial list of those findings. Certainly, identifying specific actions of teratogens, such as by their mode of action on neurotransmitters, will greatly aid us in our understanding of basic mechanisms of teratogenesis.
The data obtained so far indicate that the Drosophila assay can, in addition to being a potential tool for teratogens pre-screening, be used for understanding some of the mechanisms of teratogenesis. The vast biochemical, molecular, and developmental knowledge of Drosophila should provide us with some important clues to the roles of genetic and biochemical variable in the process of teratogenesis.
Information from the heat shock protein induction response of Drosophila has also provided us with at least some possibilities for understanding how heat shock protein induction and developmental abnormalities can be interrelated. Subsequent experiments will focus on determining whether the small heat shock proteins provide a protective function. Experiments will be carried out to determine whether cells can be protected from the effects of various teratogens. In a world that is increasingly subject to toxic compounds, understanding the mechanisms leading to teratogenesis is critical.
In 1980 she was appointed a research scientist at City of Hope Medical Center continuing her work with developing models and assays to replace in vitro teratogen testing protocols.
Appointed associate professor of biology at California State
University, San Bernar-dino in 1987, she is responsible for for
instructing
courses in molecular biology,
genetics, and developmental biology. She
continues her research on alternatives to animal use in teratogen testing
utilizing both Drosophila and human amniotic fluid, and chorionic villi
cells. Her active lab provides research experience for many graduate and
undergraduate students.
Nicole is married to an economist and
has two children,one of
who is bound to become a scientist! They spend part of their summers in Greece
enjoying non-scientific pursuits.
If you are interested in corresponding with the author, please address all correspondence to Nicole Bournias-Vardiabasis, Ph.D, CSUSB, Department of Biology, 5500 University Parkway, San Bernardino, CA 90247. Fax: 909-880-7005.
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| Copies of this journal are no longer available for sale, but our other two journals, Society & Animals and the Journal of Applied Animal Welfare Science, are available and subscriptions are quite affordable. They can be ordered online via our secure order page. |