4-Hydroxytamoxifen-induced cytotoxicity and bisphenol A: competition for estrogen receptors in human breast cancer cell lines.

Increasing concerns over the effects of environmental estrogens on wildlife and humans have highlighted the need for screening systems to assess potentially estrogenic effects of test compounds. As a result, in vitro screening methods such as cell proliferation assays using the estrogen-responsive human breast cancer cell line, MCF-7, have been developed. The present study describes an alternative in vitro approach for the assessment of such xenoestrogens, based on estrogenic rescue of MCF-7 cells from antiestrogen-induced cytotoxicity. This method measures the ability of various estrogenic compounds to compete with a known estrogen-receptor-mediated antihormonal drug, 4-hydroxytamoxifen, using the 1-[4,5-dimethylthiazol-2-yl]-3,5-diphenylformazan (MTT) assay to assess mitochondrial activity. Because 4-hydroxytamoxifen treatment of cells results in a dramatic decrease in mitochondrial dehydrogenase activity which is directly related to their estrogen-receptor content, inhibition of this effect with estrogenic compounds represents an estrogen-receptor interaction, or estrogenic rescue. The estrogenic compounds tested include a weak xenoestrogen, bisphenol A (BPA), and two biological estrogens, 17alpha- and 17beta-estradiol. Competitive inhibition of 4-hydroxytamoxifen-induced cytotoxicity by BPA was compared to that of the biological estrogens. The results indicate that the biological estrogens can successfully compete with the antiestrogen in a dose-dependent manner. In addition, the assay is sensitive enough to detect estrogenic rescue by even the very weak xenoestrogen, BPA, albeit at high BPA concentrations. This simple in vitro method could be used as an alternative or second-line screen for potential xenoestrogens.

What parents should know about estrogen-like compounds in dental materials.

The use of pit and fissure sealants has been reported to increase exposure to xenoestrogens. Because these estrogen-mimics are suspected of having many deleterious effects in animals, and perhaps humans, several types of studies were undertaken by our Biocompatibility Group. We confirmed that bisphenol A (BPA) and bisphenol A dimethacrylate (BPA-DM) have proliferative effects in cells with high levels of estrogen receptors. However, BPA was not detected by our group in American-made sealants, and BPA-DM was detectable in only a few. In addition, the surface layer of the sealant can be treated to reduce the possibility of unpolymerized BPA-DM being left on the tooth. We believe it is important to reassure parents that their children are less likely to be exposed to BPA from sealants than from the ingestion of soft drinks or canned food.

Androgens modulate interleukin-6 production by gingival fibroblasts in vitro.

BACKGROUND: Pregnancy and puberty gingivitis have been attributed to increased concentrations of circulating sex hormones. This inflammatory gingival condition is accompanied by the local production of cytokines. The aims of this in vitro study were to assess, in the presence or absence of testosterone (T) or dihydrotestosterone (DHT), the production of interleukin-6 (IL-6) by human gingival fibroblasts (hGF), and to evaluate the effects of flutamide (a common anti-androgen) in this system. METHODS: The effects of the androgens, T and DHT, on IL-6 production were measured in vitro in serum-free, phenol red-free medium. Cells were incubated with or without androgens for 72 hours; the concentration of IL-6 secreted into the medium after an additional 24-hour challenge with IL-1beta plus hormones was estimated by radioimmunoassay. The reverse transcription polymerase chain reaction was used to examine hGF and periodontal ligament cells (PDL) for the presence of androgen receptor. RESULTS: In serum-free medium, T and DHT at concentrations of 5 x 10(-8) to 10(-7)M significantly (P <0.05) inhibited IL-6 production by hGF. Flutamide, up to concentrations of 2 x 10(-5)M, did not reverse this inhibition. The androgen receptor was identified in both hGF and PDL. CONCLUSIONS: We concluded that elevated levels of androgens, specifically testosterone and dihydrotestosterone, could affect the stromal cell response to an inflammatory challenge by downregulation of IL-6 production. This in vitro study lends support to the hypothesis that increased hormones during pregnancy or puberty could modulate the development of localized inflammation.

Estrogenicity of bisphenol A and bisphenol A dimethacrylate in vitro.

Although pit and fissure sealants have been utilized extensively in dentistry as a way of preventing occlusal caries, results described by Olea et al. (1996) raised concerns about the safety of sealants and other resin-based dental materials due to the reported presence of bisphenol A (BPA) and its dimethacrylate ester (BPA-DM). Although the release of these compounds from dental materials has not been substantiated by two subsequent studies, we believed it was important to confirm or refute the report that BPA and BPA-DM have estrogenic activity in vitro. We grew breast cancer cells (MCF-7, T-47D, ZR-75-1) known to proliferate under estrogenic stimulation in phenol red-free DMEM containing human serum and concentrations of BPA or BPA-DM ranging from 10(-8)M to 5 x 10(-6)M. After 1 week, plates were harvested for crystal violet or sulforhodamine-B assays, and the optical densities of groups of treated cells were compared with values from control cells. At concentrations at or above 10(-6)M, both BPA and BPA-DM significantly increased cell proliferation (p < 0.05), comparable to the increase seen with 10(-9)M of estrogen. Flow cytometric methods demonstrated that these mitogenic effects occurred within 24 h of exposure to estrogen, BPA, or BPA-DM. The increase in DNA synthesis was analogous to that seen with estrogen stimulation. Thus, we confirmed that BPA and BPA-DM cause cell proliferation at micromolar concentrations that exceed the effective concentrations of estrogen by 1 to 10,000-fold.

Identification and characterization of estrogen-like components in commercial resin-based dental restorative materials.

Recently, resin-based dental restorative materials have been targeted as potential sources of xenoestrogens, specifically bisphenol A (BPA) and bisphenol A dimethacrylate (BAD), which could contribute to overall estrogen load and result in deleterious side effects. The present study used high-pressure liquid chromatography (HPLC) to analyze twenty-eight different commercially available dental resins for the presence of BPA and/or BAD. In addition, sublines of the MCF-7 human breast tumor cell line were cultured in the presence of eluates from eleven of the dental resins and assessed for proliferative responses using the sulforhodamine B assay. Only one resin, Delton II, had detectable levels of BPA or BAD that could be verified by Fourier transform infrared spectrometry. Likewise, eluates from Delton II were the only samples that elicited a significant proliferative response in two of the MCF-7 sublines tested. Therefore, we conclude that dental resins in general do not represent a significant source of BPA or BAD exposure.

In vitro cytotoxicity of resin-containing restorative materials after aging in artificial saliva.

Studies have reported that dental resin-based materials release substances which have biological liabilities. However, some current methods for detecting these substances may not be adequate to detect biologically relevant concentrations. In the current study, we hypothesized that resin-based materials exhibit cytotoxic effects and alter cellular function in vitro when high-pressure liquid chromatography (HPLC-UV detection) cannot detect any release of substances. We further hypothesized that this release continues even after aging the samples in artificial saliva. Five types of composite or compomer materials (Z-100, Tetric Ceram, Dyract AP, Solitaire, and Clearfil AP-X) and one organically modified ceramic material (Definite) were tested after aging in artificial saliva for 0, 7, or 14 days. Cytotoxicity was assessed using direct contact with fibroblasts and measurement of succinic dehydrogenase activity after 48 h of exposure post aging. Release of substances from the materials was assessed using HPLC with UV detection. Altered cellular function was estimated by measuring proliferation of MCF-7 cells with sulforhodamine staining. HPLC showed that whereas initial release of substances was higher without aging, this release dropped significantly after 7 or 14 days of aging, and was equivalent to the Teflon controls after 14 days for four of the materials (Tetric Ceram, Definite, Solitaire, and Clearfil AP-X). Without aging in saliva, all materials had cytotoxicities > 50% of the Teflon negative controls. After 14 days of aging, all materials except the Definite continued to show severe cytotoxicity. Only the Definite could be tested for its ability to alter cellular function because of the continuing toxicity of the other materials. This modified ceramic material caused a significant proliferative effect on the MCF-7 cells indicating that sufficient substances were released to alter cellular function. We concluded that all of these commercially available resin-based dental materials continue to release sufficient components to cause lethal effects or alter cellular function in vitro even after 2 weeks of aging in artificial saliva.

Effects of transforming growth factor-beta and platelet-derived growth factor on human gingival fibroblasts grown in serum-containing and serum-free medium.

Both transforming growth factor-beta (TGF-beta) and platelet-derived growth factor (PDGF) have been shown to affect cell proliferation in vitro. The hypothesis being tested was that the effects of the 2 cytokines would be modulated by the presence of serum in the medium. Gingival fibroblasts, obtained from periodontally healthy patients, were maintained in primary culture. Dose response experiments were performed for each growth factor in serum-free medium and in medium containing natural or heat-inactivated fetal bovine serum (10% FBS). Changes in cell numbers were quantified by crystal violet staining. The optimal concentrations of the individual factors (10 ng/ml TGF-beta1, 20 ng/ ml PDGF-BB) were then used when the 2 factors were tested in various sequences. In serum-free medium or in medium with 10% natural serum, the response to PDGF-BB was dose-dependent up to 40 ng/ml; however, with 10% heat-inactivated serum, the maximal response was seen at 20 ng/ml. The largest increase in cell numbers was produced by the simultaneous exposure to the two cytokines, rather than a sequential presentation. The findings suggest that the 48-h growth response of human gingival fibroblasts to 10 ng/ml TGF-beta1 or 20 ng/ ml PDGF-BB in serum-free medium was equivalent to growth obtained in medium containing heat-inactivated 10% FBS without added growth factors.

Human periodontal ligament and gingival fibroblast response to TGF-beta 1 stimulation.

The purpose of this study was to measure the time-sequence response of RNA and protein synthesis to transforming growth factor-beta 1 (TGF-beta 1) by human periodontal ligament (HPDLF) and gingival (HGF) fibroblasts in culture. HPDLF and HGF were cultured from explants of healthy gingival tissue and freshly extracted teeth. Cultures of 8 x 10(4) cells/ml were exposed to medium containing 3H-uridine and 35S-methionine with TGF-beta 1 at concentrations from 10(-9) M to 10(-21) M, or control medium, for up to 60 hours in order to assess RNA and protein synthesis. Protein concentrations of comparable cultures were also assayed colorimetrically. Results were reported as specific activity (CPM/microgram protein). The results indicate that 10(-9) M TGF-beta 1 treated cultures showed a significant increase in RNA synthesis by HPDLF and HGF over time, as compared to the control cultures. HPDLF showed a significant increase in protein synthesis over time while that by HGF was not significant as compared to the control cultures. Lower concentrations of TGF-beta 1 demonstrated no significant differences from control. Results suggest that the effects of TGF-beta 1 on HPDLF and HGF are both time and dose dependent, with 10(-9) M TGF-beta 1 providing the best response of those concentrations tested. These findings support the concept that TGF-beta 1 may play a role in periodontal regeneration due to its ability to promote fibroblast RNA and protein synthesis. The results also demonstrate that although these two cells types appear morphologically similar, they exhibit distinct biological responses to growth factors such as TGF-beta 1.

Role of medullary I1-imidazoline and alpha 2-adrenergic receptors in the antihypertensive responses evoked by central administration of clonidine analogs in conscious spontaneously hypertensive rats.

The antihypertensive action of clonidine analogs are generally ascribed to stimulation of medullary alpha 2-adrenergic receptors. Several recent studies have implicated the newly characterized class of imidazoline receptors as playing a more important role in this regard. In this study, a series of doses of four imidazoline and two nonimidazoline antihypertensive drugs were administered by intracisternal (i.c.) injection to freely moving spontaneously hypertensive rats. All six drugs produced a dose-dependent fall in mean arterial pressure (MAP) and heart rate (HR). The most potent agents in lowering MAP were the imidazoline agents, particularly moxonidine, a drug that has high affinity for I1-imidazoline receptors, but relatively low affinity for alpha 2-adrenergic receptors. There was a high correlation between the apparent binding affinity (Ki) for I1-receptors on membranes derived from bovine rostral ventrolateral medulla and the relative potency for lowering MAP after i.c. injection in spontaneously hypertensive rats. In contrast, no correlation existed between binding affinity values for alpha 2-adrenergic receptors derived from the same source and the antihypertensive response. The results of these experiments are consistent with a role for medullary I1-imidazoline receptors in mediating the antihypertensive action of clonidine and related imidazoline compounds in conscious hypertensive animals. The nonimidazoline agent, guanfacine, although a clinically effective antihypertensive agent, was not efficacious after i.c. injection. This discrepancy may be explained by an opposing pressor action or by a depressor action mediated by alpha 2-adrenergic receptors at an alternate site when the drug is peripherally administered. In contrast, the other nonimidazoline antihypertensive agent azepexole was fully efficacious after i.c. injection of the highest doses. Further studies may be necessary to determine whether drugs such as azepexole have a receptor target other than I1 or alpha 2-adrenergic receptors. We conclude that antihypertensive efficacy within the lower brainstem of conscious animals is predicted by interaction with I1-imidazoline but not alpha 2-adrenergic receptors.

Modulation by progesterone of interleukin-6 production by gingival fibroblasts.

The gingivitis associated with pregnancy has been attributed to increased concentrations of circulating estrogen and/or progesterone. However, the mechanism by which these steroids increase gingival inflammation is not known. Interleukin-6 (IL-6), a pleiotropic cytokine produced by many cell types including human gingival fibroblasts (hGF), is secreted in response to inflammatory challenges such as bacterial lipopolysaccharide and interleukin-1 (IL-1). This study tested the hypothesis that progesterone could modulate the local production of IL-6 by hGF. The effects of progesterone on IL-6 production were measured in vitro in serum-free, phenol red-free medium to eliminate possible effects of such medium additives. The concentration of IL-6 secreted into supernatant medium after a 24 hour challenge with IL-1 beta was estimated by radioimmunoassay. Total RNA from steroid-treated hGF was probed for IL-6 mRNA. In serum-free medium, progesterone dose-dependently and significantly (P < 0.05) inhibited IL-6 production by hGF, as did the glucocorticoids hydrocortisone (HC) and dexamethasone. At progesterone concentrations common in late pregnancy, IL-6 production was reduced to levels 40 to 50% of control. In addition, mRNA was significantly down-regulated by progesterone and HC, at both basal levels and after IL-1 beta challenge. These results suggest that high levels of progesterone during pregnancy affect the development of localized inflammation by down-regulation of IL-6 production, rendering the gingiva less efficient at resisting the inflammatory challenges produced by bacteria.

Role of prostanoids in the regulation of central cholinergic receptor sensitivity.

Tachyphylaxis develops to the hypertensive response to central (i.c.v.) injection of carbachol in conscious rats. This pressor response exhibits tachyphylaxis if the injection is repeated within 8 hr of the first injection. Blockade of brain prostaglandin synthesis with indomethacin does not inhibit the pressor response to carbachol in naive rats, but eliminates the pressor response to carbachol when the muscarinic agonist is repeated within a few hours of the first injection. If the time interval is extended to permit return of the full response (i.e., 24 hr later), indomethacin no longer inhibits the pressor response. The related cyclooygenase inhibitor meclofenamate produced effects which were identical to those of indomethacin, but at approximately 10-fold higher doses. When shorter acting drugs (duration of action < 30 min), physostigmine or arecoline, were used according to the same paradigm, indomethacin was less effective at inhibiting the pressor response to the second injection, even when the two agonist injections were spaced only 30 min apart. The ability of indomethacin to enhance central muscarinic receptor tachyphylaxis was also observed in carbachol-induced hypothermia. The density of diencephalic muscarinic receptors was estimated by using N-[3H]methylscopolamine as a probe. Carbachol-induced a down-regulation of muscarinic receptors, and indomethacin increased the extent of this down regulation. These findings suggest that prostaglandins play a role in the development of tachyphylaxis to brain muscarinic receptor stimulation: activation of prostaglandin synthesis may decelerate the development of desensitization to muscarinic agonists.

Medium flow rate modulates autocrine-paracrine feedback of GH and PRL release by perifused GH3 cells.

We previously documented both the spontaneous acceleration of growth hormone (GH) and prolactin (PRL) production by GH3 cells during perifusion and the suppression of their production during plate culture. We here present the role played by medium flow itself in this differential behavior. Increasing rates of perifusion flow (pump rates of 1 to 5 ml/h, equivalent to chamber flow rates of 0.19 to 1.3 microliters.min-1.mm-2 of cross-sectional area) were associated with enhanced GH and PRL secretion. Flow rate-dependent basal hormone secretion rates were established quickly and were stable for the first 10 to 14 h of perifusion. The previously documented independent, spontaneous, and continuously accelerating production of both hormones that followed during the subsequent 40 (PRL) to 60 (GH) h of perifusion was also shown to be flow-rate related. Any time the rate of medium flow was changed within an experiment, the rate of hormone secretion was modulated. However, that modulation did not interrupt ongoing flow-associated acceleration of hormone production once the latter had begun. In addition, GH3 cell product(s) from one cell column reversibly inhibited secretion from cells in a downstream column. The inhibition did not occur when cells in the downstream column had been exposed to trypsin. Other work had suggested that neither GH, PRL, insulinlike growth factor-I, leucine, nor nutrient exhaustion were responsible for the effect. These data are consistent with autocrine-paracrine feedback regulation of GH3 cells by a secretory product(s). Feedback would thus provide a mechanism to effect flow-rate-dependent modulation of GH and PRL release, and to explain accelerating hormone production during perifusion.

Reduction of T-kininogen messenger RNA levels by dexamethasone in the adjuvant-treated rat.

When inflammation is induced in rats following injection of Freund's complete adjuvant, steady state levels of T-I and T-II kininogen mRNAs increase markedly as do plasma levels of T-I and T-II kininogens. When rats are additionally treated with dexamethasone, T-I and T-II steady state mRNA levels and plasma levels of T-kininogens are reduced. The results suggest that dexamethasone may affect the magnitude of T-kininogen gene induction caused by inflammation.

Autocrine-paracrine inhibition of growth hormone and prolactin production by GH3 cell-conditioned medium.

In previous work we have shown that perifused GH3 cells exhibit spontaneously accelerating growth hormone (GH) and prolactin (PRL) secretory rates. This behavior contrasts with GH and PRL secretion rates that are decreasing or stable over the same 3-d period in static cell culture. We now report that GH3 cells maintained in serum-supplemented medium produce an autocrine-paracrine factor(s) which inhibits GH secretion in plate culture; PRL release is frequently reduced as well. The inhibitory effect of conditioned medium on GH secretion was concentration dependent, whereas PRL release was stimulated at low and inhibited at high concentrations over the same range. Extensive dialysis of conditioned medium using membranes with a molecular weight cut-off of 12,000-14,000 did not remove GH inhibition but produced a retentate that stimulated PRL secretion. Heat-inactivation of conditioned medium did not abolish inhibition of GH release but did remove the PRL-stimulatory effect. IGF-I added to fresh culture medium did not reproduce the GH-inhibitory effects of conditioned medium. We conclude that GH3 cell secretory behavior in perifusion and plate culture systems may be partially explained by the production of an autocrine-paracrine factor: its accumulation in plate culture inhibits GH and PRL secretion whereas its removal, by perifusing medium, allows GH and PRL secretion to accelerate.

Implementation of a self-health project by baccalaureate students.

With increasing evidence that life-style is an important influence on health, three nursing faculty members at the University of Minnesota implemented a learning project to enable students to assess, plan, and evaluate their own life-styles. The goal was to have the students attempt to make positive changes. As part of a "health concepts" nursing course, students became much more aware of social, economic, environmental, and cultural factors that either enhanced or detracted from their ability to achieve their ideal life-styles. The students responded favorably to this assignment because of the potential benefits of investing in themselves while pursuing the rigorous program leading to a nursing degree.

Deleterious effects of fungizone on growth hormone and prolactin secretion by cultured GH3 cells.

The rates at which growth hormone (GH) and prolactin (PRL) are spontaneously secreted from a rat pituitary tumor cell line (GH3) were significantly reduced when these cells were maintained in medium containing 2.5 micrograms/ml Fungizone (Fz). The reduction in hormone secretion was not immediately reversed by removal of Fz during perifusion, but after 3 wk in control medium, secretory rates approached the pre-Fz treatment levels. In plated cells, secretion of GH was reduced by Fz in a dose-dependent manner, whereas PRL secretion was significantly reduced only by the highest concentration (2.5 micrograms/ml) of Fz. We concluded that Fz is not an acceptable medium constituent for the long-term culture of GH3 cells. However, because its effects are reversible, its short-term use as a decontaminating agent might eliminate the necessity for reinitiating the culture of cells whose secretory behavior must be followed in long-term protocols.

GH3 cell secretion of growth hormone and prolactin increases spontaneously during perifusion.

UNLABELLED: GH3 cell secretory activity was studied in long-term perifusion to define previously reported spontaneous increases in growth hormone (GH) and prolactin production (PRL). Mechanically harvested cells (1 X 10(7)/column) were perifused at 4 ml/h for 72 h. A basal period of variable duration (8 to 12 h), during which hormone secretion was stable, was followed by steadily increasing secretion rates. Changes in cell number were not sufficient to account for increased hormone secretion rates: a) there was no significant change in cell count after 72 h (0.97 +/- 0.03 X 10(7); n = 18); b) mean cell column DNA content increased 25.5% above the base value, whereas GH secretion rose 385% and PRL rose 178% (n = 5). Observed differences in the duration of the basal secretion period, the basal secretory rate, and the magnitude of secretory rate increase were associated with several variables: a) variability within a subline was a function of passage number: GH secretion decreased and PRL secretion increased with subculture number; b) cells with identical lot and freeze numbers, but received at different times, behaved differently; c) the presence of an antifungal agent (nystatin) altered hormone secretion reproducibly. CONCLUSIONS: a) rates of GH and PRL secretion rise spontaneously in perifusion without a proportional increase in GH3 cell number; b) fluctuations in the rate of GH3 cell secretion of GH and PRL are not entirely random but are determined by several definable variables.

Hypothalamic and pituitary enzymatic degradation of luteinizing hormone-releasing hormone during the 4-day estrous cycle of the rat. Assessment by high-performance liquid chromatography.

Luteinizing hormone-releasing hormone (LHRH) degrading activity may be of physiological significance as a mechanism capable of partial regulation of hypothalamic LHRH release as well as LHRH levels at the gonadotroph. The possibility of cyclic fluctuations in LHRH-degrading activity was investigated in female rat hypothalami and pituitaries. These tissues were collected at selected time points during the 4-day estrous cycle, homogenized, and centrifuged at 100,000 g. Supernatants were incubated with synthetic LHRH, the reactions terminated, and the decapeptide and its products separated by high-performance liquid chromatography. Degradation of LHRH incubated with active cytosol was estimated by comparison of integrated LHRH peak area with that from incubations with heat-inactivated cytosol. Hypothalamic LHRH degradation was depressed during the latter hours of diestrus 2, a time during which the LHRH content in the hypothalamus has been reported to be increasing. From diestrus 24.00 h to proestrus 15.00 h, there was a significant increase in degrading activity. This was then followed by a decline from 15.00 to 18.00 h proestrus; at the time of the LH surge, the activity had not undergone significant increase in comparison to 18.00 h. Pituitary LHRH degradation was significantly increased during the 6-hour period preceding the surge, but was significantly depressed at the surge. The hypothalamic reduction in activity associated with diestrus 2 as well as the hypothalamic and pituitary reductions associated with proestrus may represent a permissive effect allowing increased LHRH accumulation in the hypothalamus and its prolonged action in the pituitary.(ABSTRACT TRUNCATED AT 250 WORDS)