Environmental estrogens and reproductive health: a discussion of the human and environmental data

Estrogenic activity of certain xenobiotics is an established mechanism of toxicity that can impair reproductive function in adults of either sex, lead to irreversible abnormalities when administered during development, or cause cancer. The concern has been raised that exposure to ambient levels of estrogenic xenobiotics may be having widespread adverse effects on reproductive health of humans and wildlife. The purpose of this review is to evaluate (a) the nature of the evidence supporting this concern, and (b) the adequacy of toxicity screening to detect, and risk assessment procedures to establish safe levels for, agents acting by this mechanism. Observations such as adverse developmental effects after maternal exposure to therapeutic levels of the potent estrogen diethylstilbestrol or male fertility problems after exposure to high levels of the weak estrogen chlordecone clearly demonstrate that estrogenicity is active as a toxic mechanism in humans. High level exposures to estrogenic compounds have also been shown to affect specific wildlife populations. However, there is little direct evidence to indicate that exposures to ambient levels of estrogenic xenobiotics are affecting reproductive health. Reports of historical trends showing decreasing reproductive capacity (e.g., decreased sperm production over the last 50 years) are either inconsistent with other data or have significant methodologic inadequacies that hinder interpretation. More reliable historical trend data show an increase in breast cancer rate, but the most comprehensive epidemiology study to data failed to show an association between exposure to persistent, estrogenic organochlorine compounds and breast cancer. Clearly, more work needs to be done to characterize historical trends in humans and background incidence of abnormalities in wildlife populations, and to test hypotheses about ambient exposure to environmental contaminants and toxic effects, before conclusions can be reached about the extent or possible causes of adverse effects. It is unlikely that current lab animal testing protocols are failing to detect agents with estrogenic activity, as a wide array of estrogen-responsive endpoints are measured in standard testing batteries. Routine testing for aquatic and wildlife toxicity is more limited in this respect, and work should be done to assess the validity of applying mammalian toxicology data for submammalian hazard identification. Current risk assessment methods appear to be valid for estrogenic agents, although the database for evaluating this is limited. In conclusion, estrogenicity is an important mechanism of reproductive and developmental toxicity; however, there is little evidence at this point that low level exposures constitute a human or ecologic health risk. Given the potential consequences of an undetected risk, more research is needed to investigate associations between exposures and effects, both in people and animals, and a number of research questions are identified herein. The lack of evidence demonstrating widespread xenobiotic-induced estrogenic risk suggests that far-reaching policy decisions can await these research findings.     

p107 and p130 associated cyclin A has altered substrate specificity

We demonstrate that p107 and p130 immune complexes exhibit kinase activity. We have tested such immune complexes with four substrates commonly utilized to assay Cdk activity, including all three known members of the retinoblastoma family. Immunodepletion revealed this kinase activity could be abolished by removal of either cyclin A or Cdk2 but was unaffected by removal of Cdk4 or any D-type cyclin. The appearance of p107 associated activity followed the accumulation of p107 protein. In contrast, the kinase activity associated with p130 immune complexes became apparent after mid-G1, coincident with p130 hyperphosphorylation. GST-Rb, GST-p107, and GST-p130 (where GST indicates glutathione S-transferase) were equally suitable substrates in p107 and p130 immune complex kinase assays, yielding activity equal to 25% of the cyclin A activity present. The p107 and p130 associated activity was unable to phosphorylate histone H1, suggesting the p107 and p130 associated cyclin A/Cdk2 may represent a distinct pool with a distinct substrate specificity. The p107 and p130 associated activity was released from the immune complexes upon incubation with ATP and Mg2+ and exhibited the same substrate preference observed with the untreated immune complex. Our data suggest that p107 and p130 recognize, or form by association, a distinct pool of cyclin A/Cdk2 that preferentially phosphorylates retinoblastoma family members.     

Analysis of variant forms of porcine surfactant polypeptide-C by nano-electrospray mass spectrometry

Electrospray (ES) mass spectrometry has been used to analyse preparations of porcine pulmonary surfactant polypeptide-C (SP-C). A number of variant forms of the native 35-residue dipalmitoylated peptide were detected including (a) C-terminally methylated SP-C, (b) C-terminally methylated and methionine oxidized SP-C, (c) N-terminally truncated, C-terminally methylated and methionine oxidized SP-c, (d) C-terminally elongated, C-terminally methylated and methionine oxidized SP-C, and (e) tripalmitoylated, C-terminally methylated and methionine oxidized SP-C. C-terminal methylation and methionine oxidation are probably a consequence of the sample handling procedure. The occurrence of the C-terminally elongated form of SP-C has implications for the in vivo processing of proSP-C and the Tandem mass spectrometry (MS/MS) was used to confirm the amino acid sequence of SP-C and the presence of palmitoyl groups covalently linked to the peptide. Some of the structures of the variant forms of SP-C were determined by MS/MS.     

Modifications of current properties by expression of a foreign potassium channel gene in Xenopus embryonic cells

The development of excitable cells is characterized by highly organized patterns of expression of ion channels. During the terminal differentiation of Xenopus muscle somites, potassium currents are expressed first just after Stage 15 (early-mid neurula), following a long period during which no voltage-dependent currents can be detected in any cell in the dorsal embryo. We have investigated whether early expression of a foreign delayed rectifier potassium channel may affect this endogenous pattern of electrical development. We injected the purified cRNA of the mammalian brain Shaker-like potassium channel, Kv1.1, into fertilized Xenopus eggs. The resulting currents were analyzed in blastomeres during a 12-hr period prior to Stage 15 and in differentiating muscle cells after Stage 15. In injected embryos, a high fraction of blastomeres expressed a delayed rectifier-type current. The Kv1.1 current could be distinguished from the endogenous muscle delayed potassium current (IK,X) by its very different voltage dependence. Separation of currents based on this difference indicated that, in injected embryos, IK,X appeared much earlier in development than in control embryos. Furthermore, even in cells which expressed solely Kv1.1-type current, the sensitivity of the current to dendrotoxin declined dramatically during development, approaching that of IK,X. These data suggest an interaction between Kv1.1 and endogenous channel subunits, and/or modification of the Kv1.1 protein by the embryonic cells in ways not seen in Xenopus oocytes or mammalian cell lines.     

Specific serological diagnosis of leprosy with a recombinant Mycobacterium leprae protein purified from a rapidly growing mycobacterial host

In this report we demonstrate the utility of an monoclonal antibody inhibition enzyme-linked immunosorbent assay based on the Mycobacterium leprae 35-kDa protein, purified from the rapidly growing host Mycobacterium smegmatis, for the serodiagnosis of multibacillary leprosy. The assay proved highly specific (97.5%) and sensitive (90%) and compared favorably with two other established methods routinely utilized for leprosy serodiagnosis.     

Dynamic imaging of the pancreas using real-time endoscopic ultrasonography with secretin stimulation

BACKGROUND: Tissue factor (TF) is a transmembrane glycoprotein that, after binding to factor VII/VIIa, initiates the extrinsic coagulation pathway, resulting in thrombin generation and its sequelae. Thrombin has been shown to induce TF mRNA in endothelium, monocytes, and smooth muscle cells, further perpetuating the thrombogenic cycle. This study was designed to determine the effect of specific inhibition of thrombin by recombinant hirudin (r-hirudin) on TF distribution after balloon angioplasty in the cholesterol-fed rabbit femoral artery and porcine coronary artery models. METHODS AND RESULTS: Thirty-five femoral arteries from 32 cholesterol-fed New Zealand White rabbits and 84 coronary arteries from 55 Yorkshire-Albino swine were studied by use of a recently developed in situ method of TF localization based on digoxigenin labeling of recombinant factor VIIa (Dig-VIIa), with correlative studies of TF immunoreactivity by use of anti-rabbit (AP-1) or anti-human (sTF) antibodies. At sites of balloon angioplasty in rabbit femoral or pig coronary arteries (double or single injury), TF-antibody and Dig-VIIa staining were noted in association with endothelial cells, smooth muscle cells, and foam cells and within the fibrous tissue matrix primarily of the adventitia and neointima. Staining was significantly greater after balloon angioplasty than in vessels that had not undergone angioplasty but was similar after single and double balloon injury. Animals treated with r-hirudin (rabbits, 1 mg/kg bolus plus 2-hour infusion; pigs, 1 mg/kg bolus plus 0.7 mg x kg(-1) x d(-1) infusion for 14 days with implantable pump) had diminished TF-antibody and Dig-VIIa staining 28 days after balloon angioplasty compared with controls (bolus heparin only). This effect was more prominent on the neointima and was more striking in the porcine than the rabbit model. CONCLUSIONS: TF expression, persistent 1 month after balloon angioplasty in rabbit femoral arteries and porcine coronary arteries, is attenuated by specific thrombin inhibition with hirudin. These results suggest that thrombin inhibition, in addition to its effect on acute thrombus formation and its effect on luminal narrowing by plaque in experimental animals, may result in a prolonged reduction in thrombogenicity of the restenotic plaque through this effect on TF expression.