George D. Bittner

Most Plastic Products Release Estrogenic Chemicals: A Potential Health Problem That Can Be Solved

Background: Chemicals having estrogenic activity (EA) reportedly cause many adverse health effects, especially at low (picomolar to nanomolar) doses in fetal and juvenile mammals. Objectives: We sought to determine whether commercially available plastic resins and products, including baby bottles and other products advertised as bisphenol A (BPA) free, release chemicals having EA. Methods: We used a roboticized MCF-7 cell proliferation assay, which is very sensitive, accurate, and repeatable, to quantify the EA of chemicals leached into saline or ethanol extracts of many types of commercially available plastic materials, some exposed to common-use stresses (microwaving, ultraviolet radiation, and/or autoclaving). Results: Almost all commercially available plastic products we sampled—independent of the type of resin, product, or retail source—leached chemicals having reliably detectable EA, including those advertised as BPA free. In some cases, BPA-free products released chemicals having more EA than did BPA-containing products. Conclusions: Many plastic products are mischaracterized as being EA free if extracted with only one solvent and not exposed to common-use stresses. However, we can identify existing compounds, or have developed, monomers, additives, or processing agents that have no detectable EA and have similar costs. Hence, our data suggest that EA-free plastic products exposed to common-use stresses and extracted by saline and ethanol solvents could be cost-effectively made on a commercial scale and thereby eliminate a potential health risk posed by most currently available plastic products that leach chemicals having EA into food products.

Effects of fibrinolysis on neurite growth from dorsal root ganglia cultured in two‐ and three‐dimensional fibrin gels

The mechanism of neurite penetration of three‐dimensional fibrin matrices was investigated by culturing embryonic chick dorsal root ganglia (DRGs) within fibrin gels, upon fibrin gels, and upon laminin. The length of neurites within three‐dimensional matrices of fibrin was decreased in a concentration‐dependent manner by agents that inhibited plasmin, e.g., aprotinin, or that inhibited plasminogen activation, e.g., ϵ‐aminocaproic acid (EACA), or plasminogen antiserum. In contrast, such agents increased the length of neurites growing out from DRGs cultured upon two‐dimensional substrates of fibrin and had no effect on the length of neurites growing out from DRGs cultured upon laminin. Visualization of neurites within three‐dimensional fibrin matrices demonstrated that the distance between fibrin strands was much smaller than the diameter of neurites. All these data were consistent with the hypothesis that fibrinolysis localized to the region of the neurite tip is an important mechanism for neurite penetration of a physical barrier of fibrin strands arranged in a three‐dimensional matrix.

Effects of fibrin micromorphology on neurite growth from dorsal root ganglia cultured in three-dimensional fibrin gels

The effect of fibrin matrix micromorphology on neurite growth was investigated by measuring the length of neurites growing in three-dimensional fibrin gels with well characterized micromorphologies. Dorsal root ganglia (DRGs) from 7-day chick embryos were entrapped and cultured in gels made from varying concentrations of fibrinogen (5-15 mg/mL) or calcium (2-10 mM). The length of growing neurites was measured with light videomicroscopy, and the number and diameter of fibrin fiber bundles were measured from scanning electron micrographs. An increase in fibrinogen concentration caused a decrease in the average fiber bundle thickness, an increase in the number of fiber bundles, and a marked decrease in neurite length. Gels made with different calcium concentrations had a similar range of variation in fibrin fiber bundle number or diameter, but these variations had little effect on neurite and associated nonneuronal cell outgrowth. These results provide insights into the process of neurite advance within fibrin and may be useful in the design of fibrin-based materials used for peripheral nerve regeneration. Furthermore, this study provides the first detailed experimental data on the micromorphology of fibrin matrices made from more than 5 mg/mL of fibrinogen and indicates that existing kinetic models of fibrin polymerization do not accurately predict fibrin structure at these higher concentrations.

Long-term survival of anucleate axons and its implications for nerve regeneration

Severed distal segments of nerve axons (anucleate axons) have now been reported to survive for weeks to years in representative organisms from most phyla, including the vertebrates. Among invertebrates (especially crustaceans), such long-term survival might involve transfer of proteins from adjacent intact cells to anucleate axons. In lower vertebrates and mammals, long-term survival of anucleate axons is more likely attributed to a slow turnover of axonal proteins and/or a lack of phagocytosis by macrophages or other cell types. Invertebrate anucleate axons that exhibit long-term survival are often reactivated by neurites that have grown from proximal nucleate segments. In mammals, induction of long-term survival in anucleate axons might allow more time to use artificial mechanisms to repair nerve axons by fusing the two severed halves with polyethylene glycol, a technique recently developed to fuse severed halves of myelinated axons in earthworms.

Axolemmal repair requires proteins that mediate synaptic vesicle fusion.

A damaged cell membrane is repaired by a seal that restricts entry or exit of molecules and ions to that of the level passing through an undamaged membrane. Seal formation requires elevation of intracellular Ca(2+) and, very likely, protein-mediated fusion of membranes. Ca(2+) also regulates the interaction between synaptotagmin (Syt) and syntaxin (Syx), which is thought to mediate fusion of synaptic vesicles with the axolemma, allowing transmitter release at synapses. To determine whether synaptic proteins have a role in sealing axolemmal damage, we injected squid and crayfish giant axons with an antibody that inhibits squid Syt from binding Ca(2+), or with another antibody that inhibits the Ca(2+)-dependent interaction of squid Syx with the Ca(2+)-binding domain of Syt. Axons injected with antibody to Syt did not seal, as assessed at axonal cut ends by the exclusion of extracellular hydrophilic fluorescent dye using confocal microscopy, and by the decay of extracellular injury current compared to levels measured in uninjured axons using a vibrating probe technique. In contrast, axons injected with either denatured antibody to Syt or preimmune IgG did seal. Similarly, axons injected with antibody to Syx did not seal, but did seal when injected with either denatured antibody to Syx or preimmune IgG. These results indicate an essential involvement of Syt and Syx in the repair (sealing) of severed axons. We suggest that vesicles, which accumulate and interact at the injury site, re-establish axolemmal continuity by Ca(2+)-induced fusions mediated by proteins such as those involved in neurotransmitter release.

Plasmalemmal repair of severed neurites of PC12 cells requires Ca2+ and synaptotagmin

Ca2+ and synaptotagmin (a Ca2+‐binding protein that regulates axolemmal fusion of synaptic vesicles) play essential roles in the repair of axolemmal damage in invertebrate giant axons. We now report that neurites of a rat pheochromocytoma (PC12) cell line transected and maintained in a serum medium form a dye barrier (exclude an external hydrophilic fluorescent dye) and survive for 24‐hr posttransection (based on morphology and retention of another hydrophilic dye internally loaded at 6‐hr posttransection). Some (25%) transected neurites that form a dye barrier regrow. Most (83%) neurites transected in a saline solution containing divalent cations (PBS++) also exclude entry of an externally placed hydrophilic fluorescent dye at 15‐min posttransection. In contrast, only 14 or 17% of neurites maintained in a divalent cation‐free solution (PBS=) or in PBS= + Mg2+, respectively, form a dye barrier. Neurites that do not form a dye barrier do not survive for 24 hr. When PC12 neurites are loaded with an antibody to squid synaptotagmin, most (81%) antibody‐loaded neurites do not form a dye barrier, whereas most (≥81%) neurites loaded with heat‐inactivated antibody or preimmune IgG do form a barrier. These data show that: 1) transected neurites of PC12 cells have mechanism(s) for plasmalemmal repair (dye barrier formation and survival); 2) Ca2+ is necessary for dye barrier formation, which occurs minutes after transection and is necessary for survival and regrowth; and 3) synaptotagmin is an essential mediator of barrier formation. The similarity in the requirements for plasmalemmal repair in this mammalian cell preparation with those reported previously for invertebrate axons suggests that mechanisms necessary for plasmalemmal repair have been conserved phylogenetically. J. Neurosci. Res. 62:566–573, 2000.

Degeneration and regeneration in crustacean peripheral nerves

SummaryMorphological (Figs. 3–5) and physiological (Fig. 2) data from several crustacean species kept at 19–21 °C show that isolated stumps of motor axons often survive intact for 150–250 days whereas sensory axonal segments usually degenerate within 20 days. Axonal segments of both motor and sensory axons that remain connected to their cell body generally remain functionally and morphologically normal after lesioning. No evidence was found for collateral innervation of denervated muscles from intact motor neurons supplying nearby muscle masses, although the motor nerve terminals may not have completely degenerated. Evidence is presented that motor axons specifically re-innervate their original muscle mass if such re-innervation occurs within 90 days after lesioning. Regenerating sensory and motor fibers make appropriate CNS and peripheral connections so as to re-establish correctly a peripheral reflex (Fig. 2C) found in intact animals (Wilson and Davis, 1965).

Polyethylene Glycol Rapidly Restores Axonal Integrity and Improves the Rate of Motor Behavior Recovery After Sciatic Nerve Crush Injury

The inability to rapidly (within minutes to hours) improve behavioral function after severance of peripheral nervous system axons is an ongoing clinical problem. We have previously reported that polyethylene glycol (PEG) can rapidly restore axonal integrity (PEG-fusion) between proximal and distal segments of cut- and crush-severed rat axons in vitro and in vivo. We now report that PEG-fusion not only reestablishes the integrity of crush-severed rat sciatic axons as measured by the restored conduction of compound action potentials (CAPs) and the intraaxonal diffusion of fluorescent dye across the lesion site, but also produces more rapid recovery of appropriate hindlimb motor behaviors. Improvement in recovery occurred during the first few postoperative weeks for the foot fault (FF) asymmetry test and between week 2 and week 3 for the Sciatic Functional Index (SFI) based on analysis of footprints. That is, the FF test was the more sensitive indicator of early behavioral recovery, showing significant postoperative improvement of motor behavior in PEG-treated animals at 24-48 h. In contrast, the SFI more sensitively measured longer-term postoperative behavioral recovery and deficits at 4-8 wk, perhaps reflecting the development of fine (distal) motor control. These and other data show that PEG-fusion not only rapidly restores physiological and morphological axonal continuity, but also more quickly improves behavioral recovery.

Plasmalemmal sealing of transected mammalian neurites is a gradual process mediated by Ca2+‐regulated proteins

Cultured mammalian PC12 or B104 cells do not instantaneously restore a plasmalemmal barrier (seal) after neurite transection, as measured using fluorescent dye probes of various sizes and saline solutions with different [Ca2+]o. Rather, transected cells gradually (from 15 to 60 min postseverance) exclude probes (dye molecules) of progressively smaller size. Furthermore, an inhibitor (calpeptin) of a Ca2+‐activated cysteine protease (calpain) and antibodies or toxins to a Ca2+‐regulated protein (synaptotagmin) and other membrane fusion proteins (syntaxin and synaptobrevin) inhibit plasmalemmal sealing. These data obtained using molecular probes on mammalian cell lines are consistent with previous data on invertebrate giant axons indicating that Ca2+ plays many roles in the formation, accumulation, and fusion/interaction of vesicles gradually forming a seal at a site of plasmalemmal damage.

Regeneration of giant axons in earthworms

Animals have evolved various strategies to repair neuronal tissue. For example, all regenerable axons in vertebrates11,12, z2-24 and insects 7,13,~0,25 and most sensory axons in crustaceans 2,3,19 repair axonal damage by allowing severed distal stumps to degenerate while outgrowths arise from intact proximal stumps. (In this paper, the term distal will be used to describe an axonal segment separated from its cell body whereas proximal will be used to describe an axonal process in contact with its original cell body.) These outgrowths can re-establish the original synaptic connections with varying degrees of success and specificity depending on the organism. In contrast, motor axons in crustaceans2,3,14,16 (cf. ref. l 8) and a CNS axon in an annelid 8 have been reported to regenerate by a functional reconnection or a fusion of slowly outgrowing proximal processes with the original distal stumps. Both types of repair mechanisms (regrowth of proximal stumps to target cells and axonal reconnection) are capable of restoring lost function. We have studied axonal regeneration in the earthworm CNS in an attempt to provide data on the following questions. (1) What is the specificity of reconnection when several adjacent, uniquely identifiable, axons are severed? (2) What is the mechanism of axonal regeneration for severed CNS giant axons in earthworms? In the ear thworm Lumbricus terrestris the ventral nerve cord (VNC) posterior to the subesophageal ganglion has 3 sets of paired peripheral roots in each body segment (Fig. 1). The most striking feature of the VNC is the set of 3 very large axons lying on its dorsal aspect, one medial giant axon positioned between two lateral giants of smaller diameter (Fig. 2A1). These axons are uniquely identifiable according to morphological (Fig. 2A1), physiological 4,21 and behavioral criteria. The extracellular potential generated by the medial giant axon is uniquely identifiable from the lateral giant potential in that the medial giant potential has a lower amplitude and a faster conduction velocity (Fig. 2Az). Behaviorally, tactile stimulation of the anterior 40