Scavestrogens Protect IMR 32 Cells from Oxidative Stress-Induced Cell Death

TOXICOLOGY AND APPLIED PHARMACOLOGY ARTICLE NO.

152, 49 –55 (1998)

TO988503

Scavestrogens Protect IMR 32 Cells from Oxidative Stress-Induced Cell Death D. Blum-Degen,* M. Haas,* S. Pohli,* R. Harth,* W. Ro¨mer,† M. Oettel,† P. Riederer,* and M. E. Go¨tz*,‡ *Clinical Neurochemistry, Department of Psychiatry, University of Wu¨rzburg, Wu¨rzburg, Germany; †Jenapharm GmbH & Co.KG, Jena, Germany; and ‡Karolinska Institute, Institute of Environmental Medicine, Stockholm, Sweden Received March 9, 1998; accepted June 2, 1998

al., 1994; Good et al., 1996). This hypothesis has prompted research efforts to identify compounds that might act as antioxidants, i.e., compounds that antagonize the deleterious actions of ROS on biomolecules. Concerning the central nervous system, appropriate antioxidant drugs have to be blood– brain barrier permeable and should not affect normal cellular function, such as neurotransmission. Such compounds could act by either directly scavenging ROS or triggering protective mechanisms inside the cell, resulting in an improved defense against ROS. In our studies we used the so-called scav(enger)estrogens J811 and J861, two D8,9-dehydro homologues of 17a-estradiol, first described by Oettel et al. in 1996. Scavestrogens are known to exhibit antioxidative activity by altering iron redox state and inhibiting the formation of superoxide anion radicals in vitro (Ro¨mer et al., 1997a). Furthermore, these compounds possess more potent radical-scavenging activities than the naturally occurring 17b-estradiol (Ro¨mer et al., 1997a, 1997b). In vitro, prolonged exposure of cells to hydrogen peroxide and/or iron leads to cell death. We have studied the protective potential of these scavestrogens, compared with 17a- and 17bestradiol, against Fenton reagent-mediated cell death (FeSO4, 50 mM plus H2O2, 200 mM) using cell culture.

Scavestrogens Protect IMR 32 Cells from Oxidative StressInduced Cell Death. Blum-Degen, D., Haas, M., Pohli, S., Harth, R., Ro¨mer, W., Oettel, M., Riederer, P., and Go¨tz, M. E. (1998). Toxicol. Appl. Pharmacol. 152, 49 –55. Oxidative stress is considered an important pathophysiological mechanism contributing to promote cell death in a broad variety of diseases including cardiovascular and neurodegenerative disorders. The so-called scavestrogens J811 and J861, structurally derived from 17a-estradiol, are potent radical scavengers and inhibitors of iron-induced cell damage in vitro. In this study the potential cytoprotective effects of the scavestrogens J811 and J861 against Fenton reagent-induced cell damage (50 mM FeSO4 plus 200 mM H2O2) were compared with those of 17a- and 17bestradiol. Cell viability studies using Trypan blue staining showed that estradiols and scavestrogens at concentrations ranging from 0.1 to 10 mM are able to protect IMR 32 neuroblastoma cells from Fenton-mediated death. In addition, these compounds decreased lipid peroxidation measured as thiobarbituric acid reactive substances and renormalize oxidative stress-increased intracellular glutathione levels. When given 6 h after the toxic stimulus, J811 and J861 rescued 60% of cells, whereas 17a- and 17b-estradiol were ineffective. These results suggest that the scavestrogens J811 and J861 are powerful antioxidants capable of interfering with radical-mediated cell death in diseases known to be aggravated by reactive oxygen species. Such compounds may be useful in the development of novel treatments for stroke or neurodegenerative disorders. © 1998 Academic Press Key Words: scavestrogens; steroids; Alzheimer’s disease; radicals; Fenton reagent; oxidative stress; IMR 32 neuroblastoma; glutathione

MATERIALS AND METHODS J811 (Estra-1,3,5(10),8-tetraene-3,17a-diol) and J861 (14a, 15a-methylene-estra-1,3,5(10),8-tetraene-3,17a-diol) were synthesized by Prof. Dr. S. Schwarz (Department of Chemistry, Jenapharm GmbH & Co.KG, Jena, Germany) with HPLC purity .99%. 17a- and 17b-estradiol, progesterone, pregnanolone, glutathione, and Trypan blue stain (0.4%) were purchased from Sigma (Deisenhofen, Germany). 1,1,3,3-Tetramethoxypropane was obtained from Fluka (Buchs, Switzerland). All other chemicals for biochemical analysis were obtained from Merck (Darmstadt, Germany). Stock solutions of J811, J861, 17a-estradiol, 17b-estradiol, pregnanolone, and progesterone were prepared in ethanol at concentrations of 10 mM and stored at 220°C. GSH standards were prepared in 150 mM H3PO4 at a concentration of 0.3 mg/ml and freshly diluted daily (0.3 ng/ml).

Oxidative stress, a cellular imbalance between production and elimination of reactive oxygen species (ROS, such as superoxide, hydrogen peroxide, and peroxynitrite), is considered to be of major pathophysiological relevance for a variety of pathological processes, such as cancer, diabetes, cataractogenesis, ischemia-reperfusion injury, and atherosclerosis, as well as for chronic progressive neurodegenerative diseases, such as amyotrophic lateral sclerosis (ALS), Parkinson’s disease (PD), and dementia of the Alzheimer type (DAT) (Go¨tz et

IMR 32 cell culture. IMR 32 cells (described by Tumilowicz et al., 1970) were obtained from the European Collection of Animal Cell Cultures (Salisbury, UK) at Passage Number 63. Cells were grown in Dulbecco’s modified Eagle’s medium (DMEM) containing 4.5% glucose, stabilized L-glutamine, and 25 mM HEPES (Pan Systems, Nu¨rnberg, Germany) supplemented with 49

0041-008X/98 $25.00 Copyright © 1998 by Academic Press All rights of reproduction in any form reserved.

50

BLUM-DEGEN ET AL.

10% inactivated fetal calf serum, sodium pyruvate (1.1 mM), and gentamycin (60 mg/l). Cells were incubated for 2 or 3 days in 75-cm2 plastic flasks (Falcon, Lincoln Park, NJ) at 37°C in a humidified atmosphere of 5% CO2/95% O2. Monolayer cultures were harvested by trypsinization (0.125% trypsin/1% EDTA in phosphate-buffered saline) and routinely tested for eventual contamination with mycoplasm with a commercial Mycoplasma Detection Kit (Boehringer Mannheim, Germany). For the described experiments, trypsinized cells were subcultured for 24 h in 6-cm diameter plastic dishes (Falcon) and grown to subconfluency. For this purpose we used DMEM, which contained 10% of dialyzed FCS (Pan Systems), to minimize the amount of endogenous steroids. Protective design. The steroidal compounds, freshly diluted in ethanol (0.1, 1, and 10 mM), were added to cell cultures simultaneously with Fenton reagent in fresh medium containing dialyzed FCS (10%). The vehicle volume of ethanol did not exceed 2 ml in 1 ml of medium and was also added to the control and Fenton reagent dishes, which contained no other additives. Following a 24-h incubation, cells were subsequently harvested for the described assays. Rescue design. Drugs were added to the cultures in DMEM containing 10% dialyzed FCS 6 h after the exposure to Fenton reagent. Following incubation for 18 h, cells were harvested. Determination of cell viability. Posttreatment cells were suspended in the remaining culture medium. An aliquot of 100 ml was used for counting Trypan blue (0.4% in 0.9% NaCl)-negative and -positive cells in a Fuchs–Rosenthal chamber to quantify viable and dead IMR 32 cells, respectively. Measurement of thiobarbituric acid reactive substances (TBARS). For the detection of TBARS, total cells in 45 ml medium were washed twice in PBS. Subsequently, each cell pellet was resuspended in 250 ml PBS and frozen at 270°C. One hundred microliters of cell suspension were lysed with 200 ml of sodium dodecyl sulfate (8.1% w/v) and incubated under air with thiobarbituric acid (1.5 ml 0.8% w/v) in 1.5 ml acetic acid, pH 3.5, and water (700 ml) for 1 h at 95°C according to a modified method described by Ohkawa et al. (1979) and Go¨tz et al. (1993). The clear solutions were measured in 3-ml glass cuvettes in a Perkin Elmer U.V./VIS spectrophotometer (Type 552S) at 532 nm. For quantitation, calibration curves were constructed using 1,1,3,3-tetramethoxypropane as a standard, ranging from 0.5 to 25 nmol. All measurements were performed in duplicate. Determination of intracellular GSH concentrations. GSH was determined after modifying the previously described assay of Krien et al. (1992). Four-hundred fifty microliters of cell suspension were pelleted and resuspended in 300 ml metaphosphoric acid (5%) containing 500 mM bis(2-aminoethyl)-amine-N,N,N9,N0,N0-pentaacetic acid-Ca,3Na-salt (DTPA) and stored at 270°C until analysis. Cell membranes were disrupted by means of ultrasound at 20 KHz for 20 s at 4°C (W 250 sonicator, Heinemann, Schwa¨bisch Gmu¨nd, Germany). For the separation of larger cell fragments, the cell suspension was centrifuged at 4°C for 20 min at 44,600g (Sorvall RC5C, rotor SM 24, DuPont-Sorvall, Bad Homburg). The supernatants were filtered through Ultrafree MC-cartridges (exclusion limit 5 kDa; Millipore, Eschborn, Germany) and centrifuged at 4°C for 90 min at 10,000g in a Microrapid-K centrifuge (Hettich, Tuttlingen, Germany). GSH concentrations were determined using 10 ml of the filtrates (1:10 dilution in water) injected onto a high performance liquid chromatography system (HPLC) equipped with a coulometric detector (ESA, Coulochem 5100A, Bedford, MA). The analytical cell (Model 5011) was run at 10.6 V. Separation of GSH was carried out at room temperature on a 5-mm, 300 3 4.0 mm Nucleosil C18 reversed-phase column (Bischoff, Leonberg, Germany). The mobile phase consisted of 2.5 mM sodium phosphate, pH 3.0. The flow rate was set at 1.0 ml/min. Identification of peaks was achieved by comparison with commercial standards (Sigma, Deisenhofen, Germany). Detection limit for GSH was 5 ng. Statistics. Data are displayed as means 6 SEM. Statistical differences in our model were calculated using one-way analysis of variance (ANOVA) Fisher’s Protected Least Significant Difference test. The accepted significance

TABLE 1 Cell Viability Independent experiments:

A (n 5 12)

B (n 5 8)

Fenton reagent

28.8 6 5.1**

32.7 6 5.2**

Compound added

1J811

1J861

117aestradiol

117bestradiol

10.1 mM 11 mM 110 mM

39.7 6 5.2 60.1 6 7.1* 77.5 6 12.2*

46.5 6 4.6 72.0 6 11.0* 64.8 6 7.7*

56.6 6 6.0* 65.4 6 6.6* 67.3 6 7.0*

47.5 6 4.4 53.4 6 7.4* 76.6 6 11.1*

Note. Cells were treated simultaneously with Fenton reagent and the compounds at the concentrations indicated. Values (% of control) are expressed as means 6 SEM of n independent experiments. **p # 0.0001 vs control; *p # 0.05 vs Fenton reagent (50 mM FeSO4 plus 200 mM H2O2). The absolute mean 6 SEM control value in experiments involving J811 and J861 (column A) was 816,625 6 63,713 (n 5 12). The absolute mean 6 SEM control value in experiments involving 17aand 17b-estradiol was 813,919 6 69,137 (n 5 8, B).

level was p , 0.05. Calculations were performed with the StatView program on Macintosh.

RESULTS

To optimize Fenton-mediated cell death in our cell culture system, we investigated in pilot studies cell survival with varying concentrations of iron sulfate (10 –150 mM) and H2O2 (100 –500 mM) for different incubation times (6, 24, 30, or 48 h) (data not shown). In the experiments reported herein, cells were exposed to Fenton reagent (50 mM FeSO4 plus 200 mM H2O2) for 24 h in DMEM containing 10% dialyzed fetal calf serum, which consistently reduced the amount of viable cells to 20 to 30% of controls (p , 0.001, ANOVA). The steroidal compounds were added either simultaneously (protective design) or 6 h after the toxic stimulus (rescue design). J811, J861, 17a- and 17b-estradiol significantly increased cell viability in a dose-dependent manner (Table 1) but progesterone and pregnanolone were ineffective (data not shown). Furthermore, even when added 6 h after the Fenton reagent, J811 and J861 (each 1 and 10 mM) were able to rescue IMR 32 cells from death whereas estradiols were ineffective (Fig. 1). Due to the production of ROS, cellular membrane components are very likely to undergo oxidation. Thus, we investigated the formation of lipid peroxides using a highly sensitive and improved assay for the determination of TBARS. Since the yield of TBARS was the same irrespective of the presence of air or inert gas (argon), all assays were performed under air. The protective activity of the steroidal compounds tested varied (Table 2), although all substances demonstrated some protective effect. Among the compounds tested, J811 and 17aestradiol were the most effective inhibitors of TBARS

51

SCAVESTROGENS PREVENT IMR 32 CELL DEATH

FIG. 1. Cell viability expressed as percent of control (100%). The absolute mean 6 SEM control value in experiments involving J811 and J861 was 935,685 6 91,900 (n 5 5). The absolute mean 6 SEM control value in experiments involving 17a- and 17b-estradiol was 788,130 6 40,167 (n 5 5). Six hours after addition of Fenton reagent (50 mM FeSO4 plus 200 mM H2O2) cells were treated with the compounds at the concentrations indicated. Analyses were performed 24 h following the toxic stimulus. Values are expressed as means 6 SEM of five independent experiments. *p # 0.05 vs Fenton reagent; **p # 0.0001 vs control.

formation. Even when compounds were added 6 h following Fenton reagent exposure, TBARS concentrations were reduced to control levels by J811, although the differences were not significant at p , 0.05 (Fig. 2). Since the important intracellular antioxidant GSH scavenges radicals directly, complexes iron, and serves as a cosubstrate for GSH-peroxidases, we investigated the cellular levels of GSH in surviving cells after 24 h exposure to the Fenton reagent. As Table 3 clearly indicates, a dose-dependent normalization of cellular GSH contents only occurs following the addition of J811 and J861. In comparison, 17a- and 17bestradiol show a tendency to decreased GSH levels, which however did not reach the significance level. Exposure to progesterone or pregnanolone was completely ineffective (not shown). Figure 3 (left) illustrates that only cells treated with

TABLE 2 Thiobarbituric Acid Reactive Substances (TBARS) Independent experiments:

n55

n56

Fenton reagent

86.6 6 8.0

157.7 6 34.1

Compound added

1J811

Control 10.1 mM 11 mM 110 mM

54.8 6 3.7* 46.0 6 3.0* 55.2 6 7.3* 64.4 6 6.2*

1J861

117aestradiol

117bestradiol

54.8 6 3.7* 107.5 6 8.9* 107.5 6 8.9* 76.8 6 4.9 89.3 6 10.4* 106.8 6 15.3 72.6 6 5.9 95.3 6 24.5* 124.2 6 21.0 73.0 6 6.7 86.2 6 15.4* 90.7 6 10.8*

Note. Cells were treated simultaneously with Fenton reagent and the compounds at the concentrations indicated. Values are expressed as means 6 SEM of n independent experiments. *p # 0.05 vs Fenton reagent (50 mM FeSO4 plus 200 mM H2O2).

J811 and J861 6 h following Fenton reagent exhibit GSH concentrations that were not different from the control value. The data demonstrating the effects of 17a- and 17b-estradiol when given 6 h after initiation of oxidative stress by Fenton reagent (Fig. 3 right) show no effect on GSH levels. However, since the Fenton reagent did not significantly increase the GSH content, it is difficult to judge real rescue effects. DISCUSSION

IMR 32 neuroblastoma cells are of human origin and upon differentiation express most of the proteins of cholinergic neurons (Neill et al., 1994). Since the cholinergic system is predominantly affected in early DAT, these cells are appropriate to study oxidative stress related cell death suggested to occur in DAT. Based on the phenolic unit, estrogens are potent radical scavengers (Mooradian, 1993; Ruiz-Larrea et al., 1995; Behl et al., 1995, 1997). Their lipophilic character is a prerequisite for a possible membrane interaction, while to demonstrate protective properties at least three rings of the steroid structure, such as that shown for 17b-estradiol, the naturally occurring estrogen, are required. Bulky alkyl substituents in two- and fourpositions have been introduced to estrogens to increase the antioxidant efficacy (Miller et al., 1996). The D8,9-dehydro derivatives of 17a-estradiol, J811 and J861, so-called scavestrogens (Oettel et al., 1996; Ro¨mer et al., 1997a,b), both increased IMR 32 cell viability even at doses 200-fold lower than the toxic stimulus hydrogen peroxide. Estradiols (but not progesterone or pregnanolone) also increased cell viability when given simultaneously with the toxic stimulus. However, the rescue effect mediated by natural estrogens was less pronounced than that mediated by the scavestrogens, particularly when these were given 6 h after the

52

BLUM-DEGEN ET AL.

FIG. 2. Thiobarbituric acid reactive substances (TBARS) expressed as pmol/106 cells. Six hours following Fenton reagent (50 mM FeSO4 plus 200 mM H2O2) cells were treated with the compounds at the concentrations indicated. Analyses were performed 24 h following the toxic stimulus. Values are expressed as means 6 SEM of five independent experiments (n 5 5). *p # 0.05 vs Fenton reagent.

Fenton reagent. The fact that progesterone and pregnanolone were not as effective as the other tested compounds could be attributed to the lack of a large delocalized p-electron system able to stabilize radical adducts, which in contrast, is a major characteristic of the scavestrogens (Ro¨mer et al., 1997a). In the rescue design, 0.1 mM J811 decreased the amount of TBARS to control levels, while there is no significant increase in cell survival (Fig. 1). This suggests that inhibition of lipid peroxidation might not be the only cause for increased cell viability. At present, we cannot explain why J811, but not J861, is effective in lowering TBARS levels, since the only structural difference between both compounds is a methylene group in the 14a,15a-position in J861. Differential interactions with lipids might be a possible reason for that phenomenon. These results evoked our interest in determining posttreatment GSH content to characterize the status of the intracellular defences against FeSO4 plus H2O2. Following treatment with TABLE 3 Glutathione Concentration Independent experiments

n 5 13

n57

Fenton reagent

2.28 6 0.58

1.80 6 0.24

Compound added

1J811

1J861

117aestradiol

117bestradiol

Control 10.1 mM 11 mM 110 mM

0.99 6 0.09* 1.98 6 0.33 1.46 6 0.35 1.28 6 0.26*

0.99 6 0.09* 1.82 6 0.28 1.43 6 0.31 1.27 6 0.21*

1.20 6 0.27* 1.74 6 0.32 1.51 6 0.36 1.29 6 0.23

1.20 6 0.27* 1.98 6 0.50 2.11 6 0.39 1.26 6 0.55

Note. Cells were treated simultaneously with Fenton reagent and the compounds at the concentrations indicated. Values are expressed as means 6 SEM. *p # 0.05 vs Fenton reagent (50 mM FeSO4 plus 200 mM H2O2).

Fenton reagent in combination with J811 or J861, the concentration of GSH per cell is approximately equal to that found in the control condition in both designs (Table 3 and Fig. 3). This suggests that there is either no need for a pronounced upregulation of GSH in the presence of scavestrogens or that GSH up-regulation occurs early and transitionally and is no longer elevated at the time of measurement. Interestingly, the scavestrogens are more potent in the rescue condition for normalizing GSH levels (Fig. 3) than in the protective condition (Table 3). We assume that the compounds investigated are, in part, inactivated by a direct reaction with the Fenton reagent when added simultaneously to the culture medium. After 6 h the death process is most likely completed and the H2O2 is largely metabolized. Viable cells at the 6-h time point, thus, probably face a higher effective dose than compared to the simultaneous addition. In our experiments, 0.1 mM of the protective agent appeared to be sufficient to rescue IMR 32 cells from Fenton reagent-induced cell death. That scavestrogens are able to rescue cells from death and prevent major changes in GSH content even when given 6 h after the toxic stimulus is suggestive of a different mechanism of action in addition to the direct scavenging of radicals, chelation of iron, and extra- and intracellular reaction with hydrogen peroxide (Ro¨mer et al., 1997a,b). Estradiol has been reported to be as effective as a-tocopherol in terms of inhibiting fatty acid oxidation but appears to be far more effective than a-tocopherol in terms of attenuating cholesterol oxidation (Ayres et al., 1996). Thus, it might be hypothesized that estradiol and the scavestrogens J811 and J861 interfere with the redox state of membrane proteins and lipids, thereby protecting the cell from more severe damage leading to death. In line with this hypothesis are data reporting that 17b-estradiol protects rat cortical synaptosomes from amyloid peptide Ab25–35 and FeSO4-induced membrane lipid peroxidation and prevents the oxidative stress-related impair-

SCAVESTROGENS PREVENT IMR 32 CELL DEATH

53

FIG. 3. Glutathione concentration expressed as mg GSH/106 viable cells. Six hours after the addition of Fenton reagent (50 mM FeSO4 plus 200 mM H2O2) cells were treated with the compounds at the concentrations indicated. Analyses were performed 24 h following the toxic stimulus. Values are expressed as means 6 SEM of five independent experiments (n 5 5). *p # 0.05 vs Fenton reagent.

ment of Na1/K1-ATPase activity, glutamate transport, and glucose transport (Keller et al., 1997). By preventing the impairment of transport systems that maintain ion homeostasis and energy metabolism, even excitotoxic synaptic degeneration might be forestalled. The fact that 17b-estradiol has been reported to directly inhibit the NMDA receptor (Weaver et al., 1997) further supports a role for estrogens in modulating excitotoxicity. In addition, intracellular compartments are involved in cell death mechanisms. Ab25–35 has been reported to cause an increase in mitochondrial ROS in PC 12 cells, which is enhanced by mutant presenilin-1 and suppressed by 17b-estradiol as indicated by mitochondrial transmembrane potential and energy charge/redox state (Mattson et al., 1997). Apart from the nongenomic effects that may promote survival of stressed neurons, estrogens may have effects on nuclear estrogen receptors, leading to increased expression of neurotrophins and their high-affinity receptors (Toran-Allerand, 1996), induction of calcium binding proteins (Darwish et al., 1991), and tau (Ferreira and Caceres, 1991). The epimeric 17a-estradiol and its homologues J811 and J861, which are characterized by an extremely weak “classical” genomic genital estrogenicity, are capable of affecting GSH concentrations, albeit being very poor ligands of the estrogen receptor. Our data as well as those presented by Behl et al. (1997), suggest a receptor-independent pathway of neuroprotection by estrogens. Although it cannot be excluded that other, as yet unknown, receptors are affected by those compounds. In this respect, redox-sensitive genes bearing an antioxidant response element (Jaiswal, 1994) might be an interesting target for further studies. In this study, we used up to 10 mM 17b-estradiol, which is a quite high dose in terms of endogenously occurring levels. It has to be considered that high-dose, long-term treatment with estrogens bears the risk of mammary tumors (Butterworth et al., 1997) and renal damage (Butterworth et al., 1998) due to

metabolic activation of estradiol via cytochrome P450 isoforms, leading, e.g., to the formation of catechol estrogens, their corresponding quinone species, and free radicals that might initiate copper-catalyzed oxidation of DNA (Malins et al., 1996; Seacat et al., 1997). On the other hand, estrogens attenuate neuronal injury caused by hemoglobin, chemical hypoxia, and excitatory amino acids in murine cortical cultures (Regan and Guo, 1997); increase dendritic spine density by reducing GABA neurotransmission in hippocampal neurons (Murphy et al., 1998); and enhance the outgrowth and survival of neocortical neurons in culture (17b-estradiol at 1 nM), which can be blocked by an antagonist of the NMDA, but not estrogen receptor (Diaz-Brinton et al., 1997). A role for estrogens in the prevention of DAT has been implicated by a retrospective epidemiological study, showing a dose- and duration-dependent correlation between estrogen replacement therapy and reduction in the incidence of DAT (Paganini-Hill and Henderson, 1994; Kawas et al., 1997). Estrogen use may delay the onset and decrease the risk of DAT in postmenopausal women (Tang et al., 1996) and long-term, low-dose treatment with 17b-estradiol is reported to improve cognitive functions, dementia symptoms, and daily activity skills in women with mild to moderate DAT (Ohkura et al., 1995). The cognitive decline in DAT is a consequence of nerve cell death and synapse loss in various brain regions. Intra- and extracellular deposits of filamentous proteins are neuropathological hallmarks of DAT. In vitro, the peptide Ab25–35 is neurotoxic via the production of ROS (Behl et al., 1994) and increased antioxidant enzyme activities protect cells from Ab25–35-induced toxicity (Sagara et al., 1996). Increasing the antioxidant capacity of cells by providing estrogens protect cells from Ab25–35 toxicity (Goodman et al., 1996; Green et al., 1996; Gridley et al., 1997). Recent results by Green et al. (1998) demonstrate that activation of the nuclear estrogen receptor is not necessary for the neuroprotective action of

54

BLUM-DEGEN ET AL.

estrogens against amyloid peptide; the latter is rather dependent on the extracellular GSH concentration. Such findings, together with our current demonstration of protective activities of J811 and J861 in vitro, suggest that estrogens and some of their derivatives may be useful in the development of novel therapeutic strategies for chronic progressive neurodegenerative disorders and other pathophysiological conditions where ROS are operative in accelerating the disease such as atherosclerosis, neurotrauma, stroke, Parkinson’s, and DAT. Further studies are required to define the endocrinological profile, the protective efficacy, and the safety of these compounds in different experimental conditions in vivo. ACKNOWLEDGMENTS We are indebted to the Jenapharm GmbH & Co. KG for supplying J811 and J861 as well as to Dr. V. Patchev, Dr. Adrienne Gorman, and Dr. Kay Double for critical reading of this manuscript. The work of Dr. D. Blum-Degen was financially supported by Jenapharm GmbH & Co. KG. (Jena, Germany).

Go¨tz, M. E., Dirr, A., Freyberger, A., Burger, R., and Riederer, P. (1993). The thiobarbituric acid assay reflects susceptibility to oxygen induced lipid peroxidation in vitro rather than levels of lipid hydroperoxides in vivo: A methodological approach. Neurochem. Int. 22, 255–262. Go¨tz, M. E., Ku¨nig, G., Riederer, P., and Youdim, M. B. H. (1994). Oxidative stress: Free radical production in neural degeneration. Pharmacol. Ther. 63, 37–122. Green, P. S., Gridley, K. E., and Simpkins, J. W. (1996). Estradiol protects against b-amyloid (25-35)-induced toxicity in SK-N-SH human neuroblastoma cells. Neurosci. Lett. 218, 165–168. Green, P. S., Gridley, K. E., and Simpkins, J. W. (1998) Nuclear estrogen receptor-independent neuroprotection by estratrienes—A novel interaction with glutathione. Neuroscience 84, 7–10. Gridley, K. E., Green, P. S., and Simpkins, J. W. (1997). Low concentrations of estradiol reduce b-amyloid (25-35)-induced toxicity, lipid peroxidation and glucose utilization in human SK-N-SH neuroblastoma cells. Brain Res. 778, 158 –165. Jaiswal, A. K. (1994). Antioxidant response element. Biochem. Pharmacol. 48, 439 – 444.

REFERENCES

Kawas, C., Resnick, S., Morrison, A., Brookmeyer, R., Corrada, M., Zonderman, A., Bacal, C., Donnell-Lingle, D., and Metter, E. (1997). A prospective study of estrogen replacement therapy and the risk of developing Alzheimer’s disease: The Baltimore Longitudinal Study of Aging. Neurology 48, 1517–1521.

Ayres, S., Tang, M., and Subbiah, M. T. R. (1996). Estradiol-17b as an antioxidant: Some distinct features when compared with common fat-soluble antioxidants. J. Lab. Clin. Med. 128, 367–375.

Keller, J. N., Germeyer, A., Begley, J. G., and Mattson, M. P. (1997). 17b-estradiol attenuates oxidative impairment of synaptic Na1/K1-ATPase activity, glucose transport, and glutamate transport induced by amyloid b-peptide and iron. J. Neurosci. Res. 50, 522–530.

Behl, C., Davis, J. B., Lesley, R., and Schubert, D. (1994). Hydrogen peroxide mediates amyloid-b–protein toxicity. Cell 77, 817– 827. Behl, C., Skutella, T., Lezoualc’h, F., Post, A., Widmann, M., Newton, C. J., and Holsboer, F. (1997). Neuroprotection against oxidative stress by estrogens: Structure–activity relationship. Mol. Pharm. 51, 535–541. Behl, C., Widmann, M., Trapp, T., and Holsboer, F. (1995). 17-b estradiol protects neurons from oxidative stress-induced cell death in vitro. Biochem. Biophys. Res. Commun. 216, 473– 482. Butterworth, M., Lau, S. S., and Monks, T. J. (1997). Formation of catechol estrogen glutathione conjugates and g-glutamyl transpeptidase-dependent nephrotoxicity of 17b-estradiol in the golden Syrian hamster. Carcinogenesis 18, 561–567. Butterworth, M., Lau, S. S., and Monks, T. J. (1998). 2-Hydroxy-4-glutathione-S-yl-17b-estradiol and 2-hydroxy-1-glutathione-S-yl-17b-estradiol produce oxidative stress and renal toxicity in an animal model of 17b-estradiolmediated nephrocarcinogenicity. Carcinogenesis 19, 133–139. Darwish, H., Krisinger, J., Furlow, J. D., Smith, C., Murdoch, F. E., and DeLuca, H. F. (1991). An estrogen-responsive element mediates the transcriptional regulation of calbindin D-9K gene in rat uterus. J. Biol. Chem. 266, 551–558. Diaz-Brinton, R., Tran, J., Profitt, P., and Montoya, M. (1997). 17b-estradiol enhances the outgrowth and survival of neocortical neurons in culture. Neurochem. Res. 22, 1339 –1351. Ferreira, A., and Caceres, A. (1991). Estrogen-enhanced neurite outgrowth: Evidence for a selective induction of tau and stable microtubules. J. Neurosci. 11, 392– 400. Good, P. F., Werner, P., Hsu, A., Olanow, C. W., and Perl, D. P. (1996). Evidence for neuronal oxidative damage in Alzheimer’s disease. Am. J. Pathol. 149, 21–28. Goodman, Y., Bruce, A., Cheng, B., and Mattson, M. P. (1996). Estrogens attenuate and corticosterone exacerbates excitotoxicity, oxidative injury, and amyloid-b-peptide toxicity in hippocampal neurons. J. Neurochem. 66, 1836 –1844.

Krien, P. M., Margon, V., and Kermici, M. (1992). Electrochemical determination of femtomole amounts of free reduced and oxidized glutathione. J. Chromatogr. 576, 255–261. Malins, D. C., Polissar, N. L., and Gunselman, S. J. (1996). Progression of human breast cancers to the metastatic state is linked to hydroxyl-radicalinduced DNA damage. Proc. Natl. Acad. Sci. USA 93, 2557–2563. Mattson, M. P., Robinson, N., and Guo, Q. (1997). Estrogens stabilize mitochondrial function and protect neural cells against the pro-apoptotic action of mutant presenilin-1. NeuroReport 8, 3817–3821. Miller, C. P., Jirkovsky, I., Hayhurst, D. A., and Adelman, S. J. (1996). In vitro antioxidant effects of estrogens with a hindered 3-OH function on the copper-induced oxidation of low density lipoprotein. Steroids 61, 305–308. Mooradian, A. D. (1993). Antioxidant properties of steroids. J. Steroid Biochem. Mol. Biol. 45, 509 –511. Murphy, D. D., Cole, N. B., Greenberger, V., and Segal, M. (1998). Estradiol increases dendritic spine density by reducing GABA neurotransmission in hippocampal neurons. J. Neurosci. 18, 2550 –2559. Neill, D., Hughes, D., Edwardson, J. A., Rima, B. K., and Allsop, D. (1994). Human IMR 32 neuroblastoma cells as a model cell line in Alzheimer’s disease research. J. Neurosci. Res. 39, 482– 493. Oettel, M., Ro¨mer, W., and Heller, R. (1996). The therapeutic potential of scavestrogens. Eur. J. Obstet. Gynecol. 65, 153. [Abstract] Ohkawa, H., Ohishi, N., and Yagi, K. (1979). Assay for lipid peroxides in animal tissues by thiobarbituric acid reaction. Anal. Biochem. 95, 351– 358. Ohkura, T., Isse, K., Akazawa, K., Hamamoto, M., Yaoi, Y., and Hagino, N. (1995). Long-term estrogen replacement therapy in female patients with dementia of the Alzheimer type: 7 case reports. Dementia 6, 99 –107. Paganini-Hill, A., and Henderson, V. W. (1994). Estrogen deficiency and risk of Alzheimer’s disease in women. Am. J. Epidemiol. 140, 256 –261. Regan, R. F., and Guo, Y. (1997). Estrogens attenuate neuronal injury due to

SCAVESTROGENS PREVENT IMR 32 CELL DEATH hemoglobin, chemical hypoxia, and excitatory amino acids in murine cortical cultures. Brain Res. 764, 133–140. Ro¨mer, W., Oettel, M., Droescher, P., and Schwarz, S. (1997a). Novel “scavestrogens” and their radical scavenging effects, iron-chelating, and total antioxidative activities: D8,9-dehydro derivatives of 17a-estradiol and 17bestradiol. Steroids 62, 304 –310. Ro¨mer, W., Oettel, M., Menzenbach, B., Droescher, P., and Schwarz, S. (1997b). Novel estrogens and their radical scavenging effects, iron-chelating, and total antioxidative activities: 17a-substituted analogs of D9(11)dehydro-17b-estradiol. Steroids 62, 688 – 694. Ruiz-Larrea, B., Leal, A., Martin, C., Martinez, R., and Lacort, M. (1995). Effects of estrogens on the redox chemistry of iron: A possible mechanism of the antioxidant action of estrogens. Steroids 60, 780 –783. Sagara, Y., Dargusch, R., Klier, F. G., Schubert, D., and Behl, C. (1996). Increased antioxidant enzyme activity in amyloid b protein-resistant cells. J. Neurosci. 16, 497–505.

55

Seacat, A. M., Kuppusamy, P., Zweier, J. L., and Yager, J. D. (1997). ESR identification of free radicals formed from the oxidation of catechol estrogens by Cu21. Arch. Biochem. Biophys. 347, 45–52. Tang, M. X., Jacobs, D., Stern, Y., Marder, K., Schofield, P., Gurland, B., Andrews, H., and Mayeux, R. (1996). Effect of estrogen during menopause on risk and age at onset of Alzheimer’s disease. Lancet 348, 429 – 432. Toran-Allerand, C. D. (1996). Mechanisms of estrogen action during neural development-mediation by interactions with the neurotrophins and their receptors. J. Steroid Biochem. Mol. Biol. 56, 168 –179. Tumilowicz, J. T., Nichols, W. W., Cholan, J. J., and Greene, A. E. (1970). Definition of a continuous human cell line derived from neuroblastoma. Cancer Res. 30, 2110 –2118. Weaver, C. E., Park-Chung, M., Gibbs, T. T., and Farb, D. H. (1997). 17b-estradiol protects against NMDA-induced excitotoxicity by direct inhibition of NMDA receptors. Brain Res. 761, 338 –341.