Effects of two types of energy restriction on methylation levels of adiponectin receptor 1 and leptin receptor overlapping transcript in a mouse mammary tumour virus-transforming growth factor-α breast cancer mouse model.

The role of adiponectin and leptin signalling pathways has been suggested to play important roles in the protective effects of energy restriction (ER) on mammary tumour (MT) development. To study the effects of ER on the methylation levels in adiponectin receptor 1 (AdipoR1) and leptin receptor overlapping transcript (Leprot) genes using the pyrosequencing method in mammary fat pad tissue, mouse mammary tumour virus-transforming growth factor-α (MMTV-TGF-α) female mice were randomly assigned to ad libitum (AL), chronic ER (CER, 15 % ER) or intermittent ER (3 weeks AL and 1 week 60 % ER in cyclic periods) groups at 10 weeks of age until 82 weeks of age. The methylation levels of AdipoR1 in the CER group were higher than those in the AL group at week 49/50 (P < 0·05), while the levels of methylation for AdipoR1 and Leprot genes were similar among the other groups. Also, the methylation levels at CpG2 and CpG3 regions of the promoter region of the AdipoR1 gene in the CER group were three times higher (P < 0·05), while CpG1 island of Leprot methylation was significantly lower compared with the other groups (P < 0·05). Adiponectin and leptin gene expression levels were consistent with the methylation levels. We also observed a change with ageing in methylation levels of these genes. These results indicate that different types of ER modify methylation levels of AdipoR1 and Leprot in different ways and CER had a more significant effect on methylation levels of both genes. Epigenetic regulation of these genes may play important roles in the preventive effects of ER against MT development and ageing processes.

Surfaces that Adhesively Discriminate Breast Epithelial Cell Lines and Lymphocytes in Buffer and Human Breast Milk.

We report new surface coatings that adhesively distinguish three breast epithelial cell lines (MCF-10A, MCF-7, and TMX2-28) when cell suspensions in buffer or breast milk are flowed over the coatings. We also report the selective capture of epithelial cells and rejection of Jurkat lymphocytes, with average selectivities exceeding 60 and captured cell purities often exceeding >99%. The surfaces achieve the dual goals of selective cell capture and resistance to fouling by proteins and other components of breast milk. The coatings do not rely on antibody targeting of cell surface markers but instead contain polycation chains embedded within a layer of end-tethered poly(ethylene glycol) (PEG) chains. The PEG, somewhat shielding the polycations, prevents surface fouling by proteins, nondesired cells, and other milk components, while the polycations produce electrostatic attractions that are heterogeneous on nanoscopic length scales. These electrostatic heterogeneities on the engineered coating, shown to produce curvature-selective particle capture in other studies, produce cell selectivity here. The ability of the engineered surfaces to discriminate these cell lines via an electrostatic driving force is remarkable, as the cells are of very similar surface charge as evidenced by their nearly identical ζ-potentials. The current surfaces, which likely distinguish cells based on their electrostatic surface landscape combined with other factors, adhesively distinguish cell lines that may differ only slightly in their expression of a surface marker, or cancer cells that minimally express EpCAM but which have different distributions of electrostatic charge on their surfaces. These surfaces are among the first to be documented for the compatibility of a polymer brush with human breast milk and may find use in technologies that capture cells from human breast milk or other complex fluids for cancer risk assessment.

Selective Adhesive Cell Capture without Molecular Specificity: New Surfaces Exploiting Nanoscopic Polycationic Features as Discrete Adhesive Units.

This work explored how molecularly non-specific polycationic nanoscale features on a collecting surface control kinetic and selectivity aspects of mammalian cell capture. Key principles for selective collector design were demonstrated by comparing the capture of two closely related breast cancer cell lines: MCF-7 and TMX2-28. TMX2-28 is a tamoxifen-selected clone of MCF-7. The collector was a silica surface, negatively-charged at pH 7.4, containing isolated molecules (~ 8 nm diameter) of the cationic polymer, poly(dimethyl-aminoethylmethacrylate), pDMAEMA. Important in this work is the non-selective nature of the pDMAEMA interactions with cells: pDMAEMA generally adheres negatively charged particles and cells in solution. We show here that selectivity towards cells results from collector design: this includes competition between repulsive interactions involving the negative silica and attractions to the immobilized pDMAEMA molecules, the random pDMAEMA arrangement on the surface, and the concentration of positive charge in the vicinity of the adsorbed pDMAEMA chains. The latter act as nanoscopic cationic surface patches, each weakly attracted to negatively-charged cells. Collecting surfaces engineered with an appropriate amount pDMAEMA, exposed to mixtures of MCF-7 and TMX2-28 cells preferentially captured TMX2-28 with a selectivity of 2.5. (This means that the ratio of TMX2-28 to MCF cells on the surface was 2.5 times their compositional ratio in free solution.) The ionic strength-dependence of cell capture was shown to be similar to that of silica microparticles on the same surfaces. This suggests that the mechanism of selective cell capture involves nanoscopic differences in the contact areas of the cells with the collector, allowing discrimination of closely related cell line-based small scale features of the cell surface. This work demonstrated that even without molecular specificity, selectivity for physical cell attributes produces adhesive discrimination.

PLD1 is overexpressed in an ER-negative MCF-7 cell line variant and a subset of phospho-Akt-negative breast carcinomas.

We have used a novel variant of the human oestrogen receptor (ER)-positive MCF-7 cell line, TMX2-28, as a model to study breast cancer. TMX2-28 cells show no detectable levels of mRNA or protein expression for the ER and express basal cytokeratins (CKs) 5, 14, and 17. cDNA microarray comparison between TMX2-28 and its parent cell line, MCF-7, identified 1402 differentially expressed transcripts, one of which was, phospholipase D1 (PLD1). Using real-time RT-PCR, we confirmed that PLD1 mRNA levels are 10-fold higher in TMX2-28 cells than in MCF-7 cells. We next examined PLD1 expression in human breast carcinomas. Phospholipase D1 mRNA levels were higher in breast tumours that expressed high-mRNA levels of basal CKs 5 and/or 17, but PLD1 mRNA levels were not significantly higher in ER-negative tumours. Phospholipase D1 protein was overexpressed in 10 of 42 (24%) breast tumours examined by IHC. Phospholipase D1 was overexpressed in 6 of 31 ER-positive tumours and 4 of 11 ER-negative tumours. Phospholipase D1 was overexpressed in three of the four tumours that showed high CK5/17 expression. Five PLD1-positive tumours were negative for phospho-Akt expression, but positive for phospho-mammalian target of rapamycin (mTOR) expression. The other five PLD1-positive breast tumours showed positive expression for phospho-Akt; however, only two of these cases were positive for phospho-mTOR. In this study, we report that PLD1 and phospho-mTOR are coexpressed in a subset of phospho-Akt-negative breast carcinomas.

Antiestrogenicity of clarified slurry oil and two crude oils in a human breast-cancer cell assay.

Exposure to crude oil and certain petroleum products can be a serious health hazard. Clarified slurry oil (CSO) is a complex mixture of hydrocarbons derived from the processing of crude oil, and is a known systemic and developmental toxicant, mutagen, and carcinogen. In the present study, CSO and two crude oils, Belridge heavy crude oil (BHCO) and Lost Hills light crude oil (LHLCO), were examined for their estrogenic and antiestrogenic properties in a human breast-cancer cell (MCF-7) assay. The MCF-7 focus assay is based on postconfluent cell growth and tissue restructuring, measured as the postconfluent development of multicellular nodules or foci. The mutagenicity indices of BHCO and LHLCO also were determined in a modified Ames Salmonella assay. Oil samples were prepared in dimethyl sulfoxide, resulting in extraction of virtually all of the aromatic compounds including the sulfur- and nitrogen-substituted three- to seven-ring polycyclic aromatic compounds comprising 62.2% of the CSO, 9% of the BHCO, and 2% of the LHLCO by total weight. None of the three samples was estrogenic in the MCF-7 focus assay. In contrast, all of the samples were antiestrogenic; that is, they inhibited the development of foci induced by 1 nM 17beta-estradiol (E2). The potencies of the oil samples for both antiestrogenicity and mutagenicity were correlated with the percent of polycyclic aromatic compounds they contained. Two potential mechanisms for the observed antiestrogenicity were examined. Radiometric analysis of the catabolism of [3H]E2 in MCF-7 cell cultures demonstrated that all three samples increased catabolism of E2. Results from a whole-cell estrogen-receptor (ER) binding assay suggested that metabolites of compounds in the oil samples might have competed with [3H]E2 for ER in the MCF-7 cultures. Thus the antiestrogenicity of the oil samples may occur through at least two mechanisms, increased catabolism of E2 and antagonistic binding to ER.

Expression of an estrogen receptor alpha variant protein in cell lines and tumors.

Human estrogen receptor alpha (ER) mRNA is a mixture of wild type and alternatively spliced variants. Many studies have examined the potential of ER mRNA profiles to serve as diagnostic/prognostic cancer biomarkers, but only a few have attempted to correlate ER mRNA profiles with protein expression. Representative ER mRNA pools were reproduced from the cDNAs of MCF-7 cells, a human breast tumor and human uterus and translated in a protease-free environment by reticulocyte lysates to determine relative translation efficiencies between the various ER mRNA transcripts and to facilitate identification of translated proteins. Cell line and tumor extracts were then examined for expression of the ER variant proteins identified in reticulocyte lysate translations. Each of the ER mRNA pools were translated by reticulocyte lysates into two ER proteins with molecular weights of approximately 60 and 52 kD. Western immunoblotting with various C- and N-terminal-directed, anti-ER antibodies and comparison with expressed ER protein standards established that the 52 kD protein (ERDelta7P) was translated from the predominant splice variant mRNA in each pool, which is missing exon 7. The 60 kD protein contained wild type ER sequence minus 61 C-terminal amino acids lost due to an intentional run off truncation. ERDelta7P expression was subsequently demonstrated in MCF-7 cells by Western immunoblotting with the site-directed antibodies. A protein corresponding to ERDelta7P was also detected in other ER positive breast tumor cell lines, and extracts of ER positive breast and uterine tumors. This widespread expression of ERDelta7P in vivo suggests that it may have some biological function. ERDelta7P may also affect immunohistochemical evaluation of ER positivity in tumors depending upon the level of its expression and the antibody used.

Toxaphene is antiestrogenic in a human breast-cancer cell assay.

Toxaphene is a complex mixture of chlorinated bornanes, bornenes, and bornadienes and was a heavily used insecticide in the United States until its use was restricted in 1982. There are conflicting reports regarding the potential for toxaphene to induce estrogenic responses in human and nonhuman animals. Due to the public concern over environmental estrogens, the estrogenicity of toxaphene was examined in a human breast-cancer cell assay, the MCF-7 focus assay, which is based on in vitro postconfluent cell proliferation and tissue restructuring. In this assay, 0.1-1 nM 17beta-estradiol (E2) produces maximum postconfluent proliferation and formation of multicellular nodules or foci. Toxaphene was also tested for its ability (1) to bind the estrogen receptor (ER) in a competitive binding assay using recombinant human ERalpha (rhER) and in a whole-cell competitive ER binding assay, and (2) to alter the catabolism of E2 in MCF-7 cell cultures. Results from the MCF-7 focus assay showed: (1) Toxaphene alone was not estrogenic between the concentrations of 0.5 nM and 10 microM, (2) toxaphene in binary combinations with chlordane, dieldrin, or endosulfan (alpha or beta) was not estrogenic, and (3) toxaphene was weakly antiestrogenic (it reduced the number of foci induced by 0.1 nM and 0.01 nM E2). Results from the competitive binding assays showed that (1) toxaphene alone did not bind rhER or ER in MCF-7 cells, and (2) toxaphene in binary combinations with other pesticides did not bind rhER. Results from the growth assay and radiometric analysis of E2 catabolism showed that (1) toxaphene did not alter the growth rate of MCF-7 cell cultures over 13 d, and (2) toxaphene did not alter the catabolism of E2. In conclusion, results from the MCF-7 focus assay demonstrate that toxaphene is weakly antiestrogenic rather than estrogenic.

Expression of an estrogen receptor alpha variant protein in cell lines and tumors.

Human estrogen receptor alpha (ER) mRNA is a mixture of wild type and alternatively spliced variants. Many studies have examined the potential of ER mRNA profiles to serve as diagnostic/prognostic cancer biomarkers, but only a few have attempted to correlate ER mRNA profiles with protein expression. Representative ER mRNA pools were reproduced from the cDNAs of MCF-7 cells, a human breast tumor and human uterus and translated in a protease-free environment by reticulocyte lysates to determine relative translation efficiencies between the various ER mRNA transcripts and to facilitate identification of translated proteins. Cell line and tumor extracts were then examined for expression of the ER variant proteins identified in reticulocyte lysate translations. Each of the ER mRNA pools were translated by reticulocyte lysates into two ER proteins with molecular weights of approximately 60 and 52 kD. Western immunoblotting with various C- and N-terminal-directed, anti-ER antibodies and comparison with expressed ER protein standards established that the 52 kD protein (ERDelta7P) was translated from the predominant splice variant mRNA in each pool, which is missing exon 7. The 60 kD protein contained wild type ER sequence minus 61 C-terminal amino acids lost due to an intentional run off truncation. ERDelta7P expression was subsequently demonstrated in MCF-7 cells by Western immunoblotting with the site-directed antibodies. A protein corresponding to ERDelta7P was also detected in other ER positive breast tumor cell lines, and extracts of ER positive breast and uterine tumors. This widespread expression of ERDelta7P in vivo suggests that it may have some biological function. ERDelta7P may also affect immunohistochemical evaluation of ER positivity in tumors depending upon the level of its expression and the antibody used.

Antiestrogenicity of environmental polycyclic aromatic hydrocarbons in human breast cancer cells.

The total concentration of 14 polycyclic aromatic hydrocarbons (PAHs) was determined to be 3400-fold greater in a sediment sample from an industrial site on the St. Lawrence River (SLR), NY, than in a sediment sample from a non-industrial site on the Kinderhook Creek (KC), NY. PAH fractions from extracts of the two environmental samples and two reconstituted mixtures as well as the 14 individual PAHs were examined for their toxic, estrogenic, and antiestrogenic activities using MCF-7 focus, recombinant human estrogen receptor (ER) binding, whole-cell ER binding, and 17beta-estradiol (E2) metabolism assays. PAH fractions from the KC and SLR were antiestrogenic; they significantly inhibited the formation of foci elicited in MCF-7 breast cancer cells by 1 nM E2. Eight of the 14 individual PAHs, and the reconstituted mixtures were also antiestrogenic. Results from the whole-cell ER binding assay and the radiometric analysis of E2 metabolism indicate that the PAHs detected in the KC and the SLR environmental samples induce antiestrogenic responses in metabolically intact human breast cancer cells through at least two mechanisms: one involving competition for the ER by a PAH metabolite and the other involving depletion of E2 through induction of metabolism.

2,2',6,6'-Tetrachlorobiphenyl is estrogenic in vitro and in vivo.

Polychlorinated biphenyls (PCBs) are ubiquitous environmental contaminants whose effects on biological systems depend on the number of and the positions of the chlorine substitutions. In the present study we examined the estrogenicity of the fully ortho-substituted PCB, 2,2',6,6'-tetrachlorobiphenyl (2,2',6,6'-TeCB). This PCB was chosen as the prototypical ortho-substituted PCB to test the hypothesis that ortho-substitution of a PCB with no para- or meta-chlorine-substitutions results in enhanced estrogenic activity. The results indicate that 2,2',6,6'-TeCB is estrogenic both in vitro, in the MCF-7 cell focus assay, and in vivo, in the rat uterotropic assay. The estrogenic activity elicited by the addition of 5 microM 2,2',6,6'-TeCB to the medium of MCF-7 cultures was inhibited by the estrogen receptor (ER) antagonist, LY156758, suggesting that 2,2',6,6'-TeCB or a metabolite is acting through an ER-dependent mechanism. Results from competitive binding assays using recombinant human (rh) ER indicate that 2,2',6,6'-TeCB does not bind rhERalpha or rhERbeta. A metabolite of 2,2',6,6'-TeCB, 2,2',6,6'-tetrachloro-4-biphenylol (4-OH-2,6,2',6'-TCB), does bind rhERalpha and rhERbeta and is also 10-fold more estrogenic than 2,2',6,6'-TeCB in the MCF-7 focus assay; however, this metabolite is not detected in the medium of MCF-7 cultures exposed to 2,2',6,6'-TeCB. Taken together, the results suggest that the estrogenicity observed in human breast cancer cells and the rat uterus may be due to 1) an undetected metabolite of 2,2',6,6'-TeCB binding to the ER, 2) 2,2',6,6'-TeCB binding directly to a novel form of the ER, or 3) an unknown mechanism involving the ER.

Benzo[k]fluoranthene enhancement and suppression of 17beta-estradiol catabolism in MCF-7 breast cancer cells.

It previously was shown that benzo[k]fluoranthene (BkF), a polycyclic aromatic hydrocarbon frequently detected in environmental samples, increases catabolism of 17beta estradiol (E2) in human breast cancer cells. Data in the present paper demonstrate that BkF both increases and inhibits the catabolism of E2 in MCF-7 breast cancer cells, and that the in vitro BkF increase and inhibition are dependent on the concentration of BkF and the length of the incubation period. A radiometric assay was used to investigate the catabolism of [3H]E2 after exposure to 5 concentrations of BkF for 6, 12, 24, 36, 48, 60, or 72 h. The concentration of BkF necessary for maximal increase in catabolism of E2 varied with the incubation period. At 6 h, a maximal increase was obtained with 0.01 and 0.1 microM, and at 48 h a maximal increase was obtained with 0.5 microM and 1 microM BkF. The increased rate of E2 catabolism was transient at lower concentrations of BkF but remained maximal at 72 h with 0.5 and 1 microM BkF. The highest concentration of BkF tested, 5 microM, was inhibitory at all time points. In contrast to BkF, fluoranthene (FL), another PAH frequently detected in environmental samples, did not significantly increase the catabolism of E2 at any of the concentrations or time points tested. Results showing that BkF inhibits the catabolism of E2 induced by 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) suggest that the BkF inhibition of cellular E2 catabolism is due to competition between BkF and E2 for the TCDD-induced enzymes. Overall, results from these studies demonstrate that BkF both increases and inhibits the cellular catabolism of E2, and emphasize the importance of considering time as well as concentration when conducting short-term in vitro assays.

Human health and chemical mixtures: an overview.

Unlike laboratory animals, people are rarely exposed to a single hazardous chemical. However, most of the information documenting adverse human health effects from environmental and occupational contaminants has come from studies focused on exposure to single chemicals, and there is little information available on how two or more contaminants affect humans. Most information on the effects of mixtures comes from animal systems and limited investigations of isolated human cells in culture, even though the study of mixtures in such systems has also been neglected. Two or more compounds may show additive, antagonistic, or synergistic interactions or may act on totally different systems and thus not interact. Furthermore, even a single chemical may have multiple effects and affect more than one organ system. Effects may vary with age, and metabolites may have totally different actions from the parent compound. This paper will review the variety of health effects in humans that may result from environmental contaminants and discuss how such contaminants may interact with each other. We will also present examples on how different contaminants interact from toxicologic studies of polychlorinated biphenyls performed as part of our Albany, New York, Superfund Basic Research Program project.

Lack of synergy by mixtures of weakly estrogenic hydroxylated polychlorinated biphenyls and pesticides.

We examined the estrogenicity of binary mixtures of the hydroxylated polychlorinated biphenyls (OHPCBs) 2,4,6-trichloro-4'-biphenylol (2,4,6-TCB-4'-OH) and 2,3,4,5-tetrachloro-4'-biphenylol and the pesticides endosulfan and dieldrin. The OHPCBs and pesticides were tested in both the MCF-7 focus assay and a competitive estrogen-receptor binding assay. Although the individual OHPCBs were estrogenic in both assays, there was no synergy when they were combined at various concentrations as equimolar mixtures. Of the pesticides, only endosulfan was estrogenic. Its weak estrogenicity was seen only in the MCF-7 focus assay at the highest concentration tested--10 microM. There was no synergy of the equimolar mixture of pesticides. To determine whether OHPCBs might respond synergistically when combined with the natural estrogen 17-beta-estradiol (E2), we tested various concentrations of 2,4,6-TCB-4'-OH in the MCF-7 focus assay in combination with physiologically relevant concentrations of E2. There was no synergy between 2,4,6-TCB-4'-OH and E2. Pretreatment for 3 or 7 days with 2,4,6-TCB-4'-OH had no effect on subsequent foci induced by a concentration of 2,4,6-TCB-4'-OH and E2. Although our results showing no synergy between the pesticides or the OHPCBs are in contrast to a recent report that binary mixtures of these same compounds produce synergistic responses in estrogen-sensitive assays, they are in agreement with the results from other assays showing a lack of synergy. Considering all results, it appears that synergy of these weakly estrogenic compounds acting through the estrogen receptor is unlikely.

Activity-dependent development of calcium regulation in growing motor axons.

In cultured nerve cord explants from the crayfish (Procambarus clarkii), the normal impulse activity levels of growing motor axons determine their response to Ca2+ influx. During depolarization or Ca2+ ionophore application, normally active tonic motor axons continue to grow, whereas inactive phasic motor axons retract and often degenerate. To determine the role of Ca2+ regulation in this difference, we measured the intracellular free Ca2+ concentration ([Ca2+]i) with fura-2. Growth cones from tonic axons normally had a higher [Ca2+]i than those from phasic axons. When depolarized with 60 mM K+, growth cones and neurites from phasic axons had a [Ca2+]i three to four times higher than did those from tonic axons. This difference in Ca2+ regulation includes greater Ca2+-handling capacity for growing tonic axons; the increase in [Ca2+]i produced by the Ca2+ ionophore 4-bromo-A23187 (0.25 microM) is four to five times greater in phasic than in tonic axons, and the decline in [Ca2+]i at the end of a depolarizing pulse is three to four times faster in tonic axons than phasic ones. Blocking impulses in growing tonic axons for 2-3 d with tetrodotoxin reduces their capacity to regulate [Ca2+]i. Thus, growing tonic and phasic axons have differences in Ca2+ regulation that develop as a result of their different activity levels. These activity-dependent differences in Ca2+ regulation influence axon growth and degeneration and probably influence other neuronal processes that are mediated by changes in [Ca2+]i.

A review of MHC-based mate preferences and fostering experiments in two congenic strains of mice.

We review our studies of mate choice with two MHC-congenic strains of mice. This work was stimulated by findings from Yamazaki and colleagues showing that male mice exhibited mate preferences for females whose MHC-haplotype was different from their own, while female mice exhibited either no preference or a weak preference for males of a particular MHC-haplotype (see Beauchamp et al., 1988). Since these findings were unexpected (mate choice theory predicts that females will be more selective than males), we studied the preferences of mice from two additional MHC-congenic strains to assess the generality of the previous findings. Specifically, the goals of our research were: (1) to determine the mate preferences of congenic mice with MHC-haplotypes derived from wild populations, (2) to compare the mate preferences of male and female mice in a test situation where each sex has a clear opportunity to make a choice, and (3) to estimate effects of cross-fostering on each sex.

Differential effects of depolarization on the growth of crayfish tonic and phasic motor axons in culture.

Previous studies have demonstrated neuron-specific differences in the inhibitory effects of depolarization upon neurite outgrowth. We examined whether there is a relationship between the normal impulse activity level of an axon and the effect of depolarization upon its growth. Inactive phasic motor axons and active tonic motor axons grow from crayfish abdominal nerve cord explants in culture. Depolarization of these axons with high K solutions produced greater inhibition of advancing growth cones from the phasic axons than from the tonic axons. During the period 20-40 min after the beginning of depolarization, tonic axon growth cones continued to advance, whereas phasic axon growth cones retracted. During chronic depolarization, all of the phasic axons retracted during the first day and approximately half of the phasic axons had degenerated after 4 days of depolarization. The majority of tonic axons continue to grow after 3 days of depolarization, and all of the tonic axon growth survived the 4 days of depolarization. The different responses of the growing phasic and tonic axons to depolarization appear to be Ca2+ dependent. The inhibitory effects of depolarization upon phasic axon growth were reduced by the Ca2+ channel blockers La3+ and Mg2+. Application of a Ca2+ ionophore, A23187, produces greater inhibition of phasic axon growth than tonic axon growth. This study demonstrates that depolarization produces greater inhibition of growth from inactive motor axons than from active motor axons. This is likely due to differences in Ca2+ regulation and/or sensitivity to intracellular Ca2+.

Assessment of PCB estrogenicity in a human breast cancer cell line.

Lower chlorinated, ortho substituted, non coplanar polychlorinated biphenyls (PCBs) are weakly estrogenic in rodents and in some in vitro assays. The estrogenic potency of six PCB congeners and one of their para-hydroxylated metabolites have been tested in an estrogen-responsive MCF-7 human breast-cancer cell-culture system, to evaluate the utility of this system for assessment of PCB and hydroxylated PCB estrogenic activity. This assay is based on the estrogen receptor-mediated induction of postconfluent cell proliferation. The results of the limited test series were generally consistent with, but not absolute in the requirement for ortho-chlorine substitution and para-hydroxylation for estrogenic potency, demonstrating the usefulness of the MCF-7 focus assay for estrogenic structure-activity evaluation of PCBs.

Intrinsic differences in axonal growth from crayfish fast and slow motoneurons.

The motoneurons innervating the fast and slow flexor muscles in the abdomen of crayfish form morphologically distinct motor terminals. Axons of the fast flexor (FF) motoneurons, which innervate the large (fast) flexor muscle, produce extensive motor terminal arbors with many branches. Axons of the slow flexor (SF) motoneurons, which innervate the thin (slow) flexor muscle, produce small terminal arbors with many fewer branches. To determine whether intrinsic factors contribute to these differences in terminal arbors, we compared regenerating axonal arbors from these two populations of motoneurons. We used an explant of the crayfish nerve cord in which axons from the FF and SF motoneurons regenerate on a homogeneous substrate. We found that regardless of the substrate, FF motor axons produced arbors with a greater total length and a greater number and density of branches than SF motor axons. These differences in regenerated arbors persisted in defined medium and in the absence of impulse activity, indicating that they result from intrinsic, neuron-specific factors. The greater branching of the FF motor axons may be related to differences in growth cones: growth cones of FF axons were significantly larger with more filopodia than growth cones of SF axons.