Abstracts 17

Neuroendocrine and Reproductive Effects of Pesticides

Stoker’, T.E., Goldman’, J.M., Cooper2, R.L. ‘Gamete and Early Embryogenesis Biology Branch and ‘Endocrinology Branch, Reproductive Toxicology Division, National Health and Environmental Effects Research Laboratory, United States Environmental Protection Agency, Research Triangle Park, North Carolina, USA

The concept of critical periods, or times during which certain key physiological events are required for normal reproductive function is well-established in reproductive biology. Perhaps the best studied critical periods are those present during gestational and pubertal development in which the specifically timed exposures to intrinsic hormones are necessary for sexual differentiation and normal growth and maturation of reproductive tissue. In addition, there are critical periods present during the estrous cycle of the adult female rat, when the appearance of dramatic endocrine alterations is necessary for normal ovulatory events to occur. Studies in our laboratory have demonstrated that brief exposures to certain pesticides, applied during a sensitive window for the neural regulation of ovulation, will block the preovulatory surge of LH, and thus delay ovulation and alter fertility (i.e. pregnancy outcome). To date, our studies have identified two classes of pesticides that appear to affect the neural control of ovulation though different noradrenergic mechanisms (Cooper et al., 1999). The formamidines block the hypothalamic regulation of the L,H surge by interfering with the alpha 2 NE receptor, while the dithiocarbamates inhibit the hypothalamic synthesis of norepinephrine.

It is also important to note that the same treatments extended over a period of several days before mating are without effect on ovulation and pregnancy outcome. This observation is important in the assessment of potential reproductive toxicants by demonstrating that a single exposure to environmental agents at appropriate times can adversely affect reproductive outcome. The development of tolerance following continued exposure to such substances also introduces the possibility that protocols using longer term exposure scenarios would likely miss the adverse effects noted after brief exposures.

In addition to the effects seen with these pesticides in the female, there appear to be fundamental sex differences in the sensitivity of the neuroendocrine control following exposure to these environmental compounds. For example, although we observed these effects in the female, similar studies in the adult male do not lead to a disruption of hypothalamic and pituitary control of the gonadal function. Finally, the difference in sensitivity between the male and female to these neuroendocrine toxicants indicates that subsequent work evaluating the female reproductive system may uncover a greater sensitivity to other CNS-active toxicants.

Cooper, R.L., Goldman, J.M., Stoker, T.E., 1999. Tox. Indust. Health 15, 26-36.

Growth Factors and Repair of the Central Nervous System

Ann Logan Department of Medicine, University of Birmingham, Birmingham, B1.5 2TT, UK

Because neurons have an absolute dependency on neurotrophic factors (NTF) for survival and growth, they are thought to play a pivotal role in neural plasticity after injury to the CNS. Yet, while numerous trophic factors like fibroblast growth factor (FGF2) neurotrophin-3 (NT3) and brain derived neuro- trophic factor (BDNF) have been identified, their ability to modify the injury response has been largely disappointing: neuronal survival is low and unsustained. Moreover, when neuronal recovery is observed, axonal growth from the injured neuron is often entrapped at the site of injury where the depot of

18 Abstracts

neurotrophic factor has been placed to promote regeneration. In the studies described in this presentation, using a model of optic nerve lesion, we investigated whether a strategy using gene-activated matrices (GAM) could circumvent these issues. The GAM consisted of condensed DNA immobilised in a collagen matrix and placed at the site of optic nerve injury. The plasmid DNA encoding the transgene was condensed with poly(D)lysine conjugated to recombinant FGF2, which facilitates DNA targeting to FGF receptor bearing axonal processes in the optic nerve. GAM containing 2.5-7.5 ug of DNA encoding reporter genes like green fluorescent protein (GFP) and thymidine kinase (TK) or alternatively FGF2 and a combination of FG.F2 with NT3 and BDNF were placed between the optic nerve stumps at the time of injury. Some animals received recombinant human FGF2 (2.5 ng). Six, 40 and 100 days after implantation of the GAM, PCR analyses showed that the DNA present in the GAM was retrogradely transported to retinal ganglion cell (RGC) somata in the retina and RT-PCR confirmed its mRNA expression at this site. Western blot analyses of these tissues also revealed the presence of the transgene products. Biological responses to the GAM were evaluated at 40 and 100 days after injury by measuring the number of RGC that transport dextran-rho- damine from their proximal axonal stumps, which reflected RGC survival. When genes encoding FGF2 or a combination of NTF with FGF2 were delivered with the GAM, the number of surviving neurons detected in the treated retina increased six to 30 fold over the number of cells measured in untreated animals receiving reporter genes or even recombinant FGF2. The combination of DNA encoding NTF with FGF2 was also more effective in sustaining cell survival than DNA encoding FGF2 alone. These results show that FGFZtargeted gene delivery using GAM result in sustained NTF action and that GAM approaches may be an alternative strategy to promote nerve survival and regeneration.

Fact and Fantasy or Exploding the Myth

Anthony D. Dayan a.dayan@toxic.u-net.com

Perceptions of the need for safety pharmacology investigation of biotechnology products (and many other forms of toxicity testing) still range from ‘Do nothing because they don’t cause problems’ or ‘We don’t have the right species’ to those who would apply a full range of standard techniques ‘Just in case’. In the middle lie those seeking regulatory reassurance for speedy development (‘No rejections and no surprises’) by doing whatever tests seem likely to satisfy one or another agency.

It is more useful to consider the known and likely biological actions of the substance under study and from that to devise a relevant range of studies. Considering the range of conventional rDNA products, such as cytokines, growth factors, fusion proteins, hormones, antibodies and soluble receptors, as well as vaccines, gene therapy and its vectors and grafts etc, and the purposes for which they are administered, it is an inevitable conclusion that many if not all are likely to have functional effects that will most sensitively or even most appropriately be detected by the gamut of ‘safety pharmacology’ test methods. The effects may be due to direct pharmacological actions of the substance itself, or the body’s responses to it, sometimes to a harmful excess of their intended pharmacodynamic action, and sometimes via the stimulation of antibodies or other binding proteins. As straightforward examples, consider the ability of high dose a-interferons to produce dromotropic and convulsogenic actions, of IL-I to cause capillary leakage, and of TNF-a to affect many vascular functions. At a more complex level, IgE antibodies or fragments that cross link the IgE receptor on mast cells may cause bronchostriction or full blown anaphylaxis, interleukins that stimulate the pathway of T-cell maturation to the T,2 phenotype, or factors that diminish pro-T,1 pathway may increase the tendency to allergic reactions, and so may anything that antagonises factors that divert immune responses towards the Thl route. Those consequences are likely to be best shown by functional testing. Growth factors that alter the development or regeneration of specialised cells are likely