- Email: [email protected]
The evaluation of the immunomodulating properties of ERA-63 a pharmaceutical with estrogenic activity
Toxicology Letters 180 (2008) 196–201
Contents lists available at ScienceDirect
Toxicology Letters journal homepage: www.elsevier.com/locate/toxlet
The evaluation of the immunomodulating properties of ERA-63 a pharmaceutical with estrogenic activity G.B. Janssen a,∗ , A.H. Penninks b , L.M.J. Knippels c , M. van Zijverden d , S. Spanhaak e a
Department of Toxicology and Drug Disposition, Organon, a part of Schering-Plough Corporation, P.O. Box 20, 5340 BH Oss, The Netherlands TNO Pharma, Zeist, The Netherlands c Numico Research B.V., Wageningen, The Netherlands d National Institute of Public Health and the Environment, Bilthoven, The Netherlands e Johnson & Johnson, Pharmaceutical Research & Development, Beerse, Belgium b
a r t i c l e
i n f o
Article history: Received 11 March 2008 Received in revised form 9 June 2008 Accepted 9 June 2008 Available online 17 June 2008 Keywords: Ethinyl estradiol Immunotoxicity Host resistance Listeria monocytogenes PFC Plaque-forming cell assay T cell-dependent antibody response
a b s t r a c t This paper describes studies performed with ERA-63 a low molecular weight pharmaceutical with intended immunomodulatory effects. Since this compound was also known to have estrogenic activity a non-conventional approach was taken in order to differentiate between estrogenic and non-estrogenicinduced immunomodulatory effects. EE was included not only for qualitative comparison (hazard identification) between immunomodulatory effects but also, in case of similar effects, to facilitate the extrapolation of the findings in the rat to anticipated effects in humans. After 28 days of treatment with dosages ranging from pharmacological up to clearly toxic levels for both compounds the immunotoxic potential was assessed by performing a T cell-dependent antibody response and a host resistance assay in rats. Selected ERA-63 dose levels (0.167–0.2, 1.67–2 and 16.7–20 mg/kg) were expected to have comparable estrogenic activity to respective EE dose levels (0.05, 0.5 and 5 mg/kg). General toxicity parameters reflecting estrogenic activity (i.e. decreased body- and organ weights of thymus and testis, and increased bilirubin and GGT levels) confirmed the comparable estrogenic activity for both compounds at the dose levels tested. Together with the comparable estrogen-related immune suppression (i.e. decreases in specific antibody responses and an increased susceptibility for Listeria monocytogenes infects) for both compounds, this indicates that available clinical data for EE facilitates the human risk assessment of ERA-63. © 2008 Elsevier Ireland Ltd. All rights reserved.
1. Introduction The pharmaceutical under investigation, ERA-63 (Fig. 1), showed efficacy in inflammatory models and was taken under development for possible treatment of rheumatic diseases (Dulos et al., 2006). However, the immunomodulatory effects of ERA-63 in the tested inflammatory models could potentially be due (in part) to its estrogenic activity (ER-␣ agonist). Immunomodulating effects as induced by estrogens are pleiotropic and may lead to both enhancement and suppression of many aspects of the immune response. Sex steroid hormones are important factors that contribute to the noted strong sexual dimorphism in the mammalian immune response. Males generally exhibit lower immune responses than females (Cernetich et al., 2006; Klein, 2000; Schuurs and Verheul, 1990). Conversely, females from many species have been shown to be more prone to producing immune responses against self-tissues
∗ Corresponding author. Tel.: +31 412 666192; fax: +31 412 666131. E-mail address: [email protected] (G.B. Janssen). 0378-4274/$ – see front matter © 2008 Elsevier Ireland Ltd. All rights reserved. doi:10.1016/j.toxlet.2008.06.857
and thus exhibit an increased tendency to develop autoimmune diseases in comparison to male individuals. In addition, an elevated humoral immune response (i.e. antibody production by B-cells) has been demonstrated in females. In male rats physiological levels of estrogen enhances immune response by increasing the synthesis of IgM antibodies (Myers and Petersen, 1985). The cell-mediated immune response also differs between males and females with females exhibiting a more pronounced Th2 response than males (Myers and Petersen, 1985). Although physiological levels of estrogens are immunostimulatory, supraphysiological levels appear to be immunosuppressive (Kovacs et al., 2002). Levels of estrogen during pregnancy may be 100–1000 times that of cycling females. This suppresses maternal Th1 responses in order to protect the conceptus and it attenuates the severity of certain disorders which are mediated by aberrant immune function, such as autoimmune diseases (Kovacs et al., 2002). Likewise, estrogen treatment protects female mice from experimental autoimmune encephalomyelitis by inhibiting the recruitment of T cells and macrophages into the CNS (Subramanian et al., 2003). In pregnant mice B cell lymphopoiesis appeared to
G.B. Janssen et al. / Toxicology Letters 180 (2008) 196–201
197
in the rat (Andrews et al., 2002b). Moreover, in previous in-house performed sub-acute toxicity experiments (data not shown) a dose level of 0.05 mg/kg EE (pharmacological dose) produced a slightly less decreased body weight in female Sprague–Dawley rats than a dose of 0.2 mg/kg ERA-63 in Wistar rats. Hence these dosages and 10 times multiples were selected for the present studies. Besides a qualitative comparison (hazard identification) of immunomodulatory effects the parallel testing with EE and the evaluation at estrogenic equipotent dose levels allowed also for a quantitative risk assessment in case similar results would be obtained. 2. Materials and methods 2.1. Test chemical
Fig. 1. Structures of ERA-63 and EE.
be selectively suppressed due to the increased levels of estrogen (Medina and Kincade, 1994). In rats estrogens enhance antibody production in the physiological range and suppress antibody production in the pharmacological range (Trawick and Bahr, 1986). Female mice treated with either physiological or pharmacological doses of estrogens are more susceptible to Listeria monocytogenes, Salmonella typhimurium and Toxoplasma gondii than non-treated female or males (Kita et al., 1985, 1989; Pung et al., 1985). Functional estrogen receptor expression is required for both the humoral (Erlandsson et al., 2003; Smithson et al., 1998; Thurmond et al., 2000) and the cell-mediated immune response (Huber et al., 1999; Liu et al., 2003; Maret et al., 2003). The two nuclear estrogen receptor isoforms (ER␣ and ERß) are differentially expressed according to immune cell type (Pillet et al., 2006). Mechanisms by which estrogen affect the functionality of the immune system may be (i) regulation of cytokine expression, (ii) affecting the elimination of autoreactive cells (negative and positive selection) in development organs (e.g. thymus and bone marrow) through an effect on the morphology and (iii) altering the patterns of apoptosis of T and B cells (Ahmed, 2000). In summary, the above indicates that interactions of estrogenic compounds are complex and due to the induced pleiotropic effects not easily predictable. The overall aim of the performed studies (the PFC assay and the host resistance model using L. monocytogenes) was to investigate whether ERA-63, a compound with intended immunomodulatory effects and having estrogenic activity, showed immunomodulating properties that could not be attributed to it’s estrogenic activity. Both the PFC assay (testing the T cell-dependent antibody response to SRBC) and the host resistance assay (testing the non-specific immunity towards a L. monocytogenes infection) are mentioned in the ICH S8 guideline for immunotoxicity testing of human pharmaceuticals as recommended functional immune tests to screen for potential immunotoxicity of compounds, and are expected to give sufficient data for risk assessment and risk management. Effects of test compounds in the PFC assay alone are considered to be about 80% predictive for immunotoxicity (Luster et al., 1992). Estrogenic potency was assessed mainly by the indirect parameter body weight as in toxicological experiments this parameter appeared to be a very sensitive parameter for estrogenic activity
Ethinyl estradiol (19-nor-17alpha-pregna-1,3,5[10]-trien-20-yne-3, 17beta-diol, CAS no 57-63-6, purity 100%) was originally obtained from N.V. Organon, Oss, The Netherlands. It was formulated by the Investigational Products Supply Department, Organon S.A., Riom, France, as a freeze dried preparation. After reconstitution with water for injection a suspension containing 5 mg ethinyl estradiol/mL in gelatin/mannitol 0.5%/5% (w/v) was obtained. Final dosing preparations were prepared by dilution with gelatin/mannitol 0.5%/5% (w/v). ERA-63 ((7-alpha,17-alpha)-7-methyl-3-methylene-19-norpregn-5[10]-en-20yn-17-ol, solution in ethanol) was originally obtained from N.V. Organon, Oss, The Netherlands. It was formulated by the Investigational Products Supply Department, Organon S.A., Riom, France, as a suspension containing 50 mg ERA-63/mL, gelatin/mannitol 0.5%/5% (w/v) and ethanol <2%. Final dosing preparations were prepared by dilution with gelatin/mannitol 0.5%/5% (w/v). Cyclophosphamide, i.v. lyophilisate (CPS, Product code: RVG 08058) was obtained from Asta Medica, Frankfurt/Main, Germany. A solution of 25 mg/mL (males) or 12.5 mg/mL (females) was prepared in water for injection shortly before administration. 2.2. Animals Male and female rats HSD/CpB: WU (spf-bred) obtained from Charles River Deutschland, Sulzfeld, Germany, were kept in macrolon cages with a soft wood bedding (four animals of one sex per cage, except for the control group of the plaqueforming cell assay in which five animals of the same sex were housed per cage). The rats were fed ad libitum using a standard pelleted rodent diet (RM3 (E) SQC, obtained from SDS Special Diets Services, Whitham, England). Animals were 6–8 weeks old upon arrival and 8–10 weeks at the start of dosing. Body weight range at the start of dosing was 158–201 g for females and 219–287 g for males in the plaque-forming cell assay and 135–188 g for females and 184–253 g for males in the host resistance assay. The animal room had a 12-h light/dark regime, a temperature of 21 ± 2 ◦ C, a relative humidity of 30–71%, and a rate of air exchange of approximately 10 times per hour. All procedures used in this experiment were compliant with the regional ethics regulations and approved by a local ethics committee. 2.3. Experimental design Two studies were performed each consisting of eight groups of male and female rats dosed orally with the vehicle (control group), ethinyl estradiol at a low dose of 0.05 mg/kg, a mid dose of 0.5 mg/kg and a high dose of 5 mg/kg (comprising a dose range starting at approximately the pharmacological dose up to clearly toxic levels), ERA-63 at a low dose of 0.2 mg/kg, a mid dose of 2 mg/kg and a high dose of 20 mg/kg (comprising a dose range starting at approximately the pharmacological dose up to clearly toxic levels), and cyclophosphamide at a dose level of 50 mg/kg (males in the host resistance assay received 100 mg cyclophosphamide/kg). Due to human error the dose levels ERA-63 in “Study II: host resistance assay with Listeria monocytogenes” were from the second day of dosing onwards 0.167, 1.67 and 16.7 mg/kg for the low, mid and high dose, respectively, instead of the above-mentioned dosages. The control group, the ethinyl estradiol treated groups and the ERA-63 treated groups were treated daily by gavage (1 mL/kg) for 29 days (first day of treatment is day 0). Cyclophosphamide was administered once intravenously (4 mL/kg) on day 27 in the host resistance assay and on day 24 in the plaque-forming cell assay (on both occasions the day before challenge with antigen or pathogen). 2.3.1. Study I: T cell-dependent antibody response in the plaque-forming cell (PFC) assay In the plaque-forming cell (PFC) assay eight animals/(group sex) (ten animals/sex for the control group) were dosed as indicated above. On day 24, all groups received an i.v. injection of 0.5 mL with 5 ± 1 × 108 sheep red blood cells (SRBC, Biotrading Benelux b.v., Mijdrecht, The Netherlands). Autopsy was performed on day 29. The animals were sacrificed using CO2 /O2 and spleen was collected aseptically, weighed and further processed to determine the number of plaque-forming cells, in addition, the thymus was weighed.
198
G.B. Janssen et al. / Toxicology Letters 180 (2008) 196–201
2.3.2. Study II: host resistance assay with L. monocytogenes In the host resistance assay sixteen animals/(group sex) were dosed as indicated above. All groups were divided over two subgroups (S1 and S2) each containing eight animals. All animals were infected (i.v.) with approximately 5 × 105 (as determined by means of fluorescent staining and flow cytometric counting and subsequently confirmed by plating and determination of the number CFU) L. monocytogenes bacteria (type 4b, source: National Institute of Health and Environmental Hygiene (RIVM)) in 500 l phosphate-buffered salt solution on day 28 (after the last dosing with test item). To provide the L. monocytogenes bacteria with sufficient virulence for experimental infections in the rat, the bacteria had been activated, prior to the administration, by culturing on fertilized and 10-day hard set eggs. Two and four days after infection the animals of subgroups S1 and S2, respectively, were anesthetized and killed by exsanguination from the abdominal aorta. Liver and spleen were removed aseptically, collected in sterile petri dishes, weighed and cooled on ice. The liver and spleen samples were transferred to the microbiological laboratory, and further processed for the quantitative detection of viable L. monocytogenes bacteria (see below). The culture of liver and spleen samples started on the day of removal of the organs. In addition, thymus, ovaries, and testis were removed and weighed. 2.4. Clinical observations and body weights The general condition and behavior of all animals was checked and recorded at least once daily. Body weights were determined twice weekly, on the day of CPS injection (positive control group), challenge (PFC assay), infection (host resistance assay) and at necropsy (all animals). 2.5. Clinical pathology Clinical pathology was conducted on the animals used for the in the host resistance assay. On day 25 blood samples were collected from the orbital plexus of all animals and on day 30 (day 2 post-infection) and 32 (day 4 post-infection) blood samples were collected at necropsy from the abdominal aorta from the two subgroups. K2 -EDTA was used as anticoagulant for hematology and blood clotting and lithium–heparin was used as anticoagulant for clinical biochemical parameters. The following hematological parameters were determined: hemoglobin, packed cell volume, red blood cell count, reticulocytes, total white blood cell count, differential white blood cell count, prothrombin time (PTT), thrombocyte count, mean corpuscular volume, mean corpuscular hemoglobin and mean corpuscular hemoglobin concentration. The following clinical biochemical parameters were determined: alkaline phosphatase activity (ALP), aspartate aminotransferase activity (ASAT) alanine aminotransferase activity (ALAT), ␥-glutamyl transferase activity (GGT), bilirubin (total), cholesterol (total), triglycerides, phospholipids, total protein, albumin, ratio albumin to globulin, urea, creatinine, sodium, potassium, calcium, chloride and inorganic phosphate. 2.6. Microbiological determination of L. monocytogenes in liver and spleen Liver and spleen samples were homogenized in sterile physiological salt solution (PSS). A tenfold amount of sterile PSS was added aseptically to each of the samples. These samples were then homogenized on ice during 60 s. Further decimal dilutions (10−2 , 10−3 and 10−4 ) were prepared in PSS and 0.1 mL was plated on non-selective sheep-blood-agar plates (Oxoid, Haarlem, the Netherlands) in duplicate. After addition of glycerol (final concentration 10%), the remaining homogenates were stored at −80 ◦ C. The plates were incubated under aerobic conditions for 48 h at 37 ◦ C and subsequently 24 h at room temperature. Typical L. monocytogenes colonies developed on the sheep-blood-agar plates were counted, and the number of colony forming units (CFU) per gram or total liver or spleen was calculated. Ten bacterial strains isolated from randomly selected colonies were used for L. monocytogenes conformational testing using the Microbact L12 system. 2.7. Determination of SRBC-specific plaque-forming cells in spleen cell suspensions The spleens were collected in a vial containing 5 mL RPMI medium. Single cell suspensions were prepared by gently rubbing the spleens through a 200 m steel filter by means of a blunt object. After removing the cell suspension, rinsing took place with 5 mL of RPMI, which was also collected. The numbers of spleen cells in RPMI medium were determined using an automated haematology analyser (K-800, Sysmex Toa, Kobe, Japan). Viability was assessed using an acridine orange/ethidium bromide labelling procedure. A suspension with a final viable spleen cell concentration of 10E+06 cells/mL was prepared. The plaque-forming cell (PFC) assay was performed according to the method described by Cunningham and Szenberg (1968). In short, three dilutions of the spleen cell suspensions (1, 0.5 and 0.25 × 10E+06 vital spleen cells) were tested in duplicate. The spleen cell suspensions were incubated with SRBC in the presence of (guinea pig) complement during 90 min at 37 ◦ C. Using a low magnification the formed plaques were counted and expressed as number of PFC/10E+06 spleen
cells and as number of PFC/spleen. If feasible, calculations were based on spleen cell dilutions with PFC counts between 50 and 100 PFC counted per chamber, since these counts are considered most reliable. 2.8. Statistical analysis of results The survival of L. monocytogenes as reflected by the number of CFU obtained from (liver or spleen) samples of the test subgroups were compared with the corresponding control subgroups by using analysis of (co)variance followed by Dunnet’s multiple comparison tests. CFU data were log transformed to obtain normal distributions. Differences in body and organ weights, hematological findings, total and differential white blood cell counts and clinical chemistry were analysed by analysis of (co)variance followed by Dunnet’s multiple comparison tests or by one-way analysis of variance followed by Dunnett’s multiple comparison tests. In case of inhomogeneous variances (tested by means of Bartlett’s test), or in case of non-continuous parameters: Kruskal–Wallis non-parametric analysis of variance followed by Mann–Whitney U-tests was performed. PFC data were evaluated by means of Brown–Forsythe ANOVA.
3. Results 3.1. General toxicity Most relevant changes in general toxicity parameters observed in both the T cell-dependent antibody response (plaque-forming cell (PFC) assay) and the host resistance assay with L. monocytogenes are summarized in Table 1. Males and females treated with EE or ERA-63 showed statistically significant dose-dependent decreases in body weight from day 3 onward in all dose groups, which resulted in a dose-dependent decrease in the terminal body weights (see Fig. 2) and a generally thin appearance at the mid- and high dose EE and ERA-63 treated groups. Additionally, the males at these dose levels showed severely reduced testis size. As expected EE and ERA-63 treatment also affected the hematological system, liver function, and the weights of spleen, thymus and testis. Regarding hematological changes relevant and statistically significant decreases in red blood cell count, hemoglobin concentration, packed cell volume and increased reticulocyte count were noted (data not shown). White blood cell and lymphocyte counts were decreased in the high dose groups with both compounds (Table 1) in both sexes. Indicative for changes in liver function were a dose-related increase in GGT, total bilirubin and prothrombin time (Table 1) and a dose-related decrease in albumin/globulin ratio. With a few exceptions, these changes were in all dose groups statistically significant. Lipid metabolism was also (statistically significant with a few exceptions) affected as indicated by decreased cholesterol and phospholipids at all dose groups and increased triglycerides in the low- and mid dose groups and decreased triglycerides in the high dose groups (data not shown) with both compounds. Organ weights such as, male testis, and for both sexes thymus and spleen weights were dose dependently and statistically significantly decreased in the EE and the ERA-63 treated groups of both sexes (Table 1). 3.2. T cell-dependent antibody response as measured by plaque-forming cell (PFC) assay Results of the relevant parameters of the PFC assay are summarized in Table 2 and the number of PFC per spleen is depicted in Fig. 3. In line with the decreased spleen weight, the number of viable cells per spleen was dose dependently decreased in both sexes (except the low dose females) with both test compounds. Regarding the numbers of PFC cells per spleen especially in males statistically significant decreases of around 50% were noted for both EE and ERA-63 at all dose levels tested. Females showed less pronounced
G.B. Janssen et al. / Toxicology Letters 180 (2008) 196–201
199
Table 1 General toxicity parameters in male and female Wistar rats after treatment for 29 days with 0 mg/kg (control), 0.05 mg/kg, 0.5 mg/kg or 5 mg/kg ethinyl estradiol (EE), or 0.167 mg/kg, 1.67 mg/kg or 16.7 mg/kg ERA-63 (mean results and standard deviation) Parameter
Control
Ethinyl estradiol (mg/kg)
0 mg/kg
0.05
ERA-63 (mg/kg)
0.5
5
0.167
1.67
16.7
Males BW day 28 (g) BW day 28 (g) P PTT (s) WBC (109 cells/L) Lymphocytes (109 cells/L) GGT (U/L) Bilirubin (mol/L) Thymus (g) P Spleen (g) P Testis (g) P
332.6 330.6 41.6 14.1 12.8 0.1 0.9 0.588 0.823 3.35
± ± ± ± ± ± ± ± ± ±
5.2 6.8 0.4 0.4 0.4 0.1 0.1 0.023 0.025 0.06
270.6 267.1 44.9 15.6 14.5 0.1 2.4 0.407 0.700 2.88
± ± ± ± ± ± ± ± ± ±
4.6** 6.4** 0.7$$$ 0.8 0.8 0.0 0.2$$$ 0.023$$$ 0.019* 0.12**
216.3 212.8 48.7 14.3 13.3 0.8 4.1 .270 0.554 0.62
± ± ± ± ± ± ± ± ± ±
4.4** 6.2** 0.4$$$ 0.5 0.5 0.1$$$ 0.3$$$ 0.032$$$ 0.033** 0.02**
177.1 194.2 50.9 12.6 11.5 5.6 7.8 0.171 0.565 0.57
± ± ± ± ± ± ± ± ± ±
5.4** 9.8** 1.1$$$ 0.6 0.5 0.5$$$ 0.5$$$ 0.016$$$ 0.018** 0.02**
236.2 230.8 48.5 15.7 14.8 0.1 2.7 0.345 0.549 2.45
± ± ± ± ± ± ± ± ± ±
4.4** 2.1** 0.7$$$ 0.6 0.6 0.0 0.2$$$ 0.020 0.015** 0.11**
208.4 201.8 49.5 14.2 13.0 0.5 4.1 0.229 0.516 0.59
± ± ± ± ± ± ± ± ± ±
5.3** 6.2** 0.9$$$ 0.6 0.5 0.1$$ 0.3$$$ 0.030$$$ 0.024** 0.03**
177.0 181.4 48.5 11.7 10.5 2.3 6.6 0.129 0.463 0.74
± ± ± ± ± ± ± ± ± ±
4.6** 5.5** 0.7$$$ 0.6* 0.6* 0.3$$$ 0.6$$$ 0.006$$$ 0.051** 0.11**
Females BW day 28 (g) BW day 28 (g) P PTT (s) WBC (109 cells/L) Lymphocytes (109 cells/L) GGT (U/L) Bilirubin (mol/L) Thymus (g) P Spleen (g) P
200.4 211.5 40.0 11.1 10.5 1.1 0.8 0.390 0.563
± ± ± ± ± ± ± ± ±
4.1 13.6 0.9 0.6 0.5 0.1 0.4 0.022 0.016
181.1 193.8 46.6 13.1 12.2 1.4 1.5 0.385 0.556
± ± ± ± ± ± ± ± ±
2.6** 8.7** 0.7** 0.7 0.7 0.1 0.4$$ 0.015 0.012
151.9 158.4 48.7 11.8 10.9 2.6 2.6 0.224 0.517
± ± ± ± ± ± ± ± ±
2.8** 8.6** 1.0** 0.7 0.6 0.2$$$ 0.3$$$ 0.014** 0.024
129.1 147.9 51.5 8.7 7.3 5.5 6.3 0.132 0.475
± ± ± ± ± ± ± ± ±
2.1** 6.5** 1.2** 0.7* 0.6** 0.6$$$ 0.7$$$ 0.011** 0.012**
185.3 196.2 41.8 12.8 11.8 1.3 0.4 0.427 0.583
± ± ± ± ± ± ± ± ±
2.3** 3.6* 0.9 0.6 0.6 0.1 0.1 0.037 0.028
150.3 171.2 48.7 12.7 11.5 1.3 4.1 0.246 0.429
± ± ± ± ± ± ± ± ±
3.2** 4.6** 1.0** 0.8 0.7 0.1 0.8$$$ 0.017** 0.015*
133.8 141.1 52.7 8.0 6.7 4.5 5.4 0.104 0.425
± ± ± ± ± ± ± ± ±
2.8** 3.4** 1.1** 0.6** 0.5** 0.5$$$ 0.6$$$ 0.009** 0.014**
Statistics homogenous data: one-way analysis of variance followed by Dunnett’s multiple comparison test (*, p < 0.05; **, p < 0.01), inhomogeneous data: Kruskal–Wallis non-parametric analysis of variance followed by Mann–Whitney U-test ($, p < 0.05; $$, p < 0.02; $$$, p < 0.002). P = determined in plaque-forming cell assay with dosages ERA-63 of 0.2, 2.0 and 20 mg/kg instead of mentioned dosages.
Fig. 3. Mean number of plaque-forming cells (PFC) per spleen in the PFC assay after treatment for 29 days with 0 mg/kg (control), 0.05 mg/kg, 0.5 mg/kg or 5 mg/kg ethinyl estradiol (EE) or 0 mg/kg (control), 0.167 mg/kg, 1.67 mg/kg or 16.7 mg/kg ERA-63 in male and female Wistar rats.
Fig. 2. Mean body weights after treatment for 29 days with 0 mg/kg (control), 0.05 mg/kg, 0.5 mg/kg or 5 mg/kg ethinyl estradiol (EE) or 0 mg/kg (control), 0.167 mg/kg, 1.67 mg/kg or 16.7 mg/kg ERA-63 in male and female Wistar rats.
Table 2 Plaque-forming cell (PFC) parameters in male and female Wistar rats after treatment for 29 days with 0 mg/kg (control), 0.05 mg/kg, 0.5 mg/kg or 5 mg/kg ethinyl estradiol (EE), or 0.2 mg/kg, 2.0 mg/kg or 20 mg/kg ERA-63 (mean results and standard deviation) Parameter
Values
Ethinyl estradiol (mg/kg)
0 mg/kg
0.05
ERA-63 (mg/kg)
0.5
5
0.2
2.0
20
Males Viability (%) Viable cells/spleen (×108 ) PFC per 106 cells PFC per spleen (×108 )
66.8 4.2 8079 3363
± ± ± ±
5.3 1.2 4099 1771
61.9 3.2 5257 1528
± ± ± ±
4.8 0.8 3030 762*
61.0 2.2 7739 1834
± ± ± ±
3.1 0.5** 5271 1607
58.6 2.0 10,319 1947
± ± ± ±
3.5 0.6** 4872 830
68.6 3.4 5413 1741
± ± ± ±
3.2 0.8 2803 1021*
67.0 2.6 6439 1669
± ± ± ±
5.0 0.6** 2549 713*
65.1 2.3 8450 2025
± ± ± ±
6.3 0.5** 4276 1214
Females Viability (%) Viable cells/spleen (×108 ) PFC per E106 cells PFC per spleen (×108 )
67.1 3.4 8717 2918
± ± ± ±
6.8 0.8 3530 1362
69.3 3.4 8560 2956
± ± ± ±
3.2 0.6 4793 1750
65.1 2.9 5688 1713
± ± ± ±
2.8 0.7 2458 947
63.6 2.1 10,844 2216
± ± ± ±
3.7 0.4** 5281 1061
64.3 3.2 9464 2746
± ± ± ±
6.6 1.0 3484 980
58.3 2.2 9811 2144
± ± ± ±
4.2 0.4* 2298 679
56.8 1.8 11,435 1989
± ± ± ±
4.2 0.4** 3187 492
Statistics homogenous data: one-way analysis of variance followed by Dunnett’s multiple comparison test (*, p < 0.05; **, p < 0.01), inhomogeneous data: Brown-Forsythe one-way analysis of variance (*, p < 0.05; ** p < 0.01).
200
G.B. Janssen et al. / Toxicology Letters 180 (2008) 196–201
treated with ERA-63 the number of CFU per organ was decreased 4 days post-infection as compared to the mid dose ERA-63 group and the high dose EE group, however this effect may be biased because the values of the high dose group was based on only four surviving animals. In spleen the pathogen burden for control animals and animals treated with low doses of EE and ERA-63 was 4 days post-infection lower or comparable to the pathogen burden 2 days post-infection. Especially in males treated with the higher doses of EE or ERA63 dose-related increases in the numbers of CFU per spleen were noted, which were statistically significant at the highest dose. In female animals these increases were much less pronounced and not statistically significant. In the high dose ERA-63 treated females the number of CFU per spleen was decreased 4 days postinfection as compared to the mid dose ERA-63 and the high dose EE groups. As remarked for the liver findings this effect may be biased because the value of the high dose group was based on only four surviving animals. The positive control group treated with CPS (data not shown) showed mortality on day 31 (4/8 males) and day 32 (2/8 females). The remaining animals that were sacrificed showed statistically significant increased numbers of viable L. monocytogenes bacteria recovered from liver and spleen. Four days after infection the number of viable L. monocytogenes bacteria recovered from liver and spleen was increased as compared to day 2 of infection. These results signify the validity of the infection model. Fig. 4. Mean number of colony forming units (CFU) per total liver and spleen in the host resistance assay with Listeria monocytogenes after treatment for 29 days with 0 mg/kg (control), 0.05 mg/kg, 0.5 mg/kg or 5 mg/kg ethinyl estradiol (EE) or 0 mg/kg (control), 0.2 mg/kg, 2 mg/kg or 20 mg/kg ERA-63 in male and female Wistar rats.
reductions at the mid and the high dose levels as is reflected by the magnitude of effects (around 30%, not statistically significant). Both for males and females a plateau effects was reached already from the low- and mid dose, respectively. Due to the overall decreases in spleen cell numbers, the number of PFC per 106 spleen cells tended to go up at the high dose levels tested. Animals treated with the positive control compound, CPS, showed as expected statistically significantly decreased thymus and spleen weights and spleen cell numbers. The number of PFC per spleen or per 106 spleen cells was negligible, indicating that CPS as positive control already demonstrated the functionality of the test system. 3.3. Host resistance assay with L. monocytogenes The number of colony forming units (CFU) per total spleen and liver, as determined in the host resistance assay with L. monocytogenes are depicted in Fig. 4a and b, respectively. Two days after infection (i.e. day 30) 1/16 females of the mid dose group EE as well as 4/16 males and 4/16 females of the high dose group ERA-63 died. From the remaining animals 1/7 males died of the high dose group ERA-63, 3 days post-infection and 4 days post-infection 1/8 males of the mid dose group ERA-63 and 1/5 females of the high dose ERA-63 died. In general, with the exception of the control group for males, the pathogen burden in the liver was higher 4 days post-infection as compared to 2 days post-infection, independent of treatment. Both EE and ERA-63 treatment especially at the higher dose levels showed dose-related increased numbers of CFU per organ 2 and 4 days post-infection. Apart from a difference in the high dose females at day 4 post-infection, the effects of ERA-63 as compared to EE showed no large quantitative differences in the numbers of CFU’s invoked by either the EE or ERA-63 treatment. In females
4. Discussion In order to facilitate a proper risk assessment of ERA-63 the overall aim of the performed studies was to investigate whether ERA-63, a compound with estrogenic activity in rats, showed additional immunomodulatory effects besides those due to its estrogenic activity. As will be discussed below, a comparison with EE at estrogenic equipotent dose levels showed neither qualitative nor quantitative differences between immunomodulating effects of the compounds. This indicates that for human risk assessment it is expected that ERA-63 treatment would not pose a different risk on an immunotoxic insult as compared to EE at equipotent dose levels. A number of parameters showed clear dose-related effects for both EE and ERA-63 and were considered to reflect estrogenic activity in rats based on literature data. These parameters were: body weight, thymus and testis organ weight, bilirubin and GGT levels. Changes in body weight are a direct consequence of an estrogenic effect on overall metabolism (Dagnault and Richard, 1997). Reduced thymus weight is related to estrogen-induced thymic atrophy (Yao and Hou, 2004) and reduced testis weight is in accordance with the known activity of estrogens acting as functional antiandrogens (antagonistic for androgens) in the male rat (Andrews et al., 2002a). Another well-known effect of EE in rats is induction of cholestasis (Azer et al., 1995), which is reflected amongst others by increased bilirubin and GGT levels. In the host resistance study the results of a number of the abovementioned parameters might have been confounded by the (degree of) infection. Therefore, the clinical pathology parameters determined pre-infection in the host resistance assay and the organ weights obtained from the PFC assay were used to compare the estrogenic effects of ERA-63 and EE. Calculation of dose–response curves for the parameters reflecting estrogenic activity allowed determination of the estrogenic potency of ERA-63 as compared to EE. As expected, based on the effects observed, the estrogenic potencies for the different dose groups treated with ERA-63 and EE were more or less comparable. However, there was some variability
G.B. Janssen et al. / Toxicology Letters 180 (2008) 196–201
depending the parameter investigated and the dose level considered. For some parameters (e.g. bilirubine and GGT) the estrogenic potency at the high dose ERA-63 was lower than the high dose EE. However, for the stronger estrogen-related parameters, e.g. body weight, growth, and thymus weight, the estrogenic potency of the high dose ERA-63 was around two times higher than the high dose EE. When considering results of the PFC both compounds showed more or less similar effects. Regarding the host resistance assays it appeared that effects of ERA-63 were, although similar, more pronounced as reflected by a higher mortality at the high dose level. However, this can be explained by the calculated higher potency of ERA-63 as compared to EE at the high dose level for some parameters. Overall, based on the similarities between noted effects of both compounds, the results demonstrated that apart from a quantitative difference there is no qualitative difference between ERA-63 and EE. Thus with respect to risk assessment this compound may be compared to EE with regard to anticipated effects on the human immune system. Although more pronounced in males, both compounds reduce the T cell-dependent antibody response in both sexes. The reduced IgM response against SRBC in the suprapharmacological range of EE is in line with findings described by others (Myers and Petersen, 1985). The dose-dependent increases in the number of viable L. monocytogenes bacteria recovered from spleen and from liver (per gram and per organ) both in the ERA-63 and EE treated groups indicate an increased sensitivity to infection. This finding is in line with similar findings in mice treated with estrogenic compounds (Pung et al., 1985). It is concluded that ERA-63 show immunomodulatory effects comparable to those of EE, and as earlier described for estrogens, i.e. decreases in specific antibody responses and an increased susceptibility for L. monocytogenes infects. Comparison of ERA-63 with EE at equipotent dose levels showed neither qualitative nor quantitative differences between the compounds. This indicates that for human risk assessment it is expected that ERA-63 administered to humans would not pose a different risk on an immunotoxic insult as compared to EE. Any anticipated effect in humans is likely to be similar to what is known with respect to equipotent EE levels. Conflict of interest None. References Ahmed, S.A., 2000. The immune system as a potential target for environmental estrogens (endocrine disrupters): a new emerging field. Toxicology 150, 191–206. Andrews, P., Freyberger, A., Hartmann, E., Eiben, R., Loof, I., Schmidt, U., Temerowski, M., Folkerts, A., Stahl, B., Kayser, M., 2002 a. Sensitive detection of the endocrine effects of the estrogen analogue ethinylestradiol using a modified enhanced subacute rat study protocol (OECD Test Guideline no. 407). Arch. Toxicol. 76, 194–202. Andrews, P., Freyberger, A., Hartmann, E., Eiben, R., Loof, I., Schmidt, U., Temerowski, M., Folkerts, A., Stahl, B., Kayser, M., 2002 b. Sensitive detection of the endocrine
201
effects of the estrogen analogue ethinylestradiol using a modified enhanced subacute rat study protocol (OECD Test Guideline no. 407). Arch. Toxicol. 76, 194–202. Azer, S.A., Canfield, P.J., Stacey, N.H., 1995. Hepatoprotection in ethinylestradioltreated rats is provided by tauroursodeoxycholic acid, but not by ursodeoxycholic acid. J. Gastroenterol. Hepatol. 10, 261–269. Cernetich, A., Garver, L.S., Jedlicka, A.E., Klein, P.W., Kumar, N., Scott, A.L., Klein, S.L., 2006. Involvement of gonadal steroids and gamma interferon in sex differences in response to blood-stage malaria infection. Infect. Immun. 74, 3190–3203. Cunningham, A.J., Szenberg, A., 1968. Further improvements in the plaque technique for detecting single antibody-forming cells. Immunology 14, 599–600. Dagnault, A., Richard, D., 1997. Involvement of the medial preoptic area in the anorectic action of estrogens. Am. J. Physiol. 272, R311–R317. Dulos, J., Hofstra, C.L., Joosten, L.A.B., Veening-Griffioen, D.H., Lucassen, M.A., Doorn, C.M., Graaf, J.H., Miltenburg, A.M.M., Dijcks, F.A., Ederveen, T.H., Boots, A.M.H., 2006. A selective estrogen receptor alpha agonist (Org 37663) suppresses inflammation and arthritis in mouse models. Ann. Rheum. Dis. 65 (suppl. II), 128. Erlandsson, M.C., Jonsson, C.A., Islander, U., Ohlsson, C., Carlsten, H., 2003. Oestrogen receptor specificity in oestradiol-mediated effects on B lymphopoiesis and immunoglobulin production in male mice. Immunology 108, 346–351. Huber, S.A., Kupperman, J., Newell, M.K., 1999. Estradiol prevents and testosterone promotes Fas-dependent apoptosis in CD4+ Th2 cells by altering Bcl 2 expression. Lupus 8, 384–387. Kita, E., Takahashi, S., Yasui, K., Kashiba, S., 1985. Effect of estrogen (17 betaestradiol) on the susceptibility of mice to disseminated gonococcal infection. Infect. Immun. 49, 238–243. Kita, E., Yagyu, Y., Nishikawa, F., Hamuro, A., Oku, D., Emoto, M., Katsui, N., Kashiba, S., 1989. Alterations of host resistance to mouse typhoid infection by sex hormones. J. Leukoc. Biol. 46, 538–546. Klein, S.L., 2000. The effects of hormones on sex differences in infection: from genes to behavior. Neurosci. Biobehav. Rev. 24, 627–638. Kovacs, E.J., Messingham, K.A., Gregory, M.S., 2002. Estrogen regulation of immune responses after injury. Mol. Cell. Endocrinol. 193, 129–135. Liu, H.B., Loo, K.K., Palaszynski, K., Ashouri, J., Lubahn, D.B., Voskuhl, R.R., 2003. Estrogen receptor alpha mediates estrogen’s immune protection in autoimmune disease. J. Immunol. 171, 6936–6940. Luster, M.I., Portier, C., Pait, D.G., White Jr., K.L., Gennings, C., Munson, A.E., Rosenthal, G.J., 1992. Risk assessment in immunotoxicology. I. Sensitivity and predictability of immune tests. Fundam. Appl. Toxicol. 18, 200–210. Maret, A., Coudert, J.D., Garidou, L., Foucras, G., Gourdy, P., Krust, A., Dupont, S., Chambon, P., Druet, P., Bayard, F., Guery, J.C., 2003. Estradiol enhances primary antigen-specific CD4 T cell responses and Th1 development in vivo. Essential role of estrogen receptor alpha expression in hematopoietic cells. Eur. J. Immunol. 33, 512–521. Medina, K.L., Kincade, P.W., 1994. Pregnancy-related steroids are potential negative regulators of B lymphopoiesis. Proc. Natl. Acad. Sci. U. S. A. 91, 5382–5386. Myers, M.J., Petersen, B.H., 1985. Estradiol induced alterations of the immune system. I. Enhancement of IgM production. Int. J. Immunopharmacol. 7, 207–213. Pillet, S., D’Elia, M., Bernier, J., Bouquegneau, J.M., Fournier, M., Cyr, D.G., 2006. Immunomodulatory effects of estradiol and cadmium in adult female rats. Toxicol. Sci. 92, 423–432. Pung, O.J., Tucker, A.N., Vore, S.J., Luster, M.I., 1985. Influence of estrogen on host resistance: increased susceptibility of mice to Listeria monocytogenes correlates with depressed production of interleukin 2. Infect. Immun. 50, 91–96. Schuurs, A.H., Verheul, H.A., 1990. Effects of gender and sex steroids on the immune response. J. Steroid Biochem. 35, 157–172. Smithson, G., Couse, J.F., Lubahn, D.B., Korach, K.S., Kincade, P.W., 1998. The role of estrogen receptors and androgen receptors in sex steroid regulation of B lymphopoiesis. J. Immunol. 161, 27–34. Subramanian, S., Matejuk, A., Zamora, A., Vandenbark, A.A., Offner, H., 2003. Oral feeding with ethinyl estradiol suppresses and treats experimental autoimmune encephalomyelitis in SJL mice and inhibits the recruitment of inflammatory cells into the central nervous system. J. Immunol. 170, 1548–1555. Thurmond, T.S., Murante, F.G., Staples, J.E., Silverstone, A.E., Korach, K.S., Gasiewicz, T.A., 2000. Role of estrogen receptor alpha in hematopoietic stem cell development and B lymphocyte maturation in the male mouse. Endocrinology 141, 2309–2318. Trawick, D.R., Bahr, J.M., 1986. Modulation of the primary and secondary antifluoresceyl antibody response in rats by 17 beta-estradiol. Endocrinology 118, 2324–2330. Yao, G., Hou, Y., 2004. Thymic atrophy via estrogen-induced apoptosis is related to Fas/FasL pathway. Int. Immunopharmacol. 4, 213–221.
Contents lists available at ScienceDirect
Toxicology Letters journal homepage: www.elsevier.com/locate/toxlet
The evaluation of the immunomodulating properties of ERA-63 a pharmaceutical with estrogenic activity G.B. Janssen a,∗ , A.H. Penninks b , L.M.J. Knippels c , M. van Zijverden d , S. Spanhaak e a
Department of Toxicology and Drug Disposition, Organon, a part of Schering-Plough Corporation, P.O. Box 20, 5340 BH Oss, The Netherlands TNO Pharma, Zeist, The Netherlands c Numico Research B.V., Wageningen, The Netherlands d National Institute of Public Health and the Environment, Bilthoven, The Netherlands e Johnson & Johnson, Pharmaceutical Research & Development, Beerse, Belgium b
a r t i c l e
i n f o
Article history: Received 11 March 2008 Received in revised form 9 June 2008 Accepted 9 June 2008 Available online 17 June 2008 Keywords: Ethinyl estradiol Immunotoxicity Host resistance Listeria monocytogenes PFC Plaque-forming cell assay T cell-dependent antibody response
a b s t r a c t This paper describes studies performed with ERA-63 a low molecular weight pharmaceutical with intended immunomodulatory effects. Since this compound was also known to have estrogenic activity a non-conventional approach was taken in order to differentiate between estrogenic and non-estrogenicinduced immunomodulatory effects. EE was included not only for qualitative comparison (hazard identification) between immunomodulatory effects but also, in case of similar effects, to facilitate the extrapolation of the findings in the rat to anticipated effects in humans. After 28 days of treatment with dosages ranging from pharmacological up to clearly toxic levels for both compounds the immunotoxic potential was assessed by performing a T cell-dependent antibody response and a host resistance assay in rats. Selected ERA-63 dose levels (0.167–0.2, 1.67–2 and 16.7–20 mg/kg) were expected to have comparable estrogenic activity to respective EE dose levels (0.05, 0.5 and 5 mg/kg). General toxicity parameters reflecting estrogenic activity (i.e. decreased body- and organ weights of thymus and testis, and increased bilirubin and GGT levels) confirmed the comparable estrogenic activity for both compounds at the dose levels tested. Together with the comparable estrogen-related immune suppression (i.e. decreases in specific antibody responses and an increased susceptibility for Listeria monocytogenes infects) for both compounds, this indicates that available clinical data for EE facilitates the human risk assessment of ERA-63. © 2008 Elsevier Ireland Ltd. All rights reserved.
1. Introduction The pharmaceutical under investigation, ERA-63 (Fig. 1), showed efficacy in inflammatory models and was taken under development for possible treatment of rheumatic diseases (Dulos et al., 2006). However, the immunomodulatory effects of ERA-63 in the tested inflammatory models could potentially be due (in part) to its estrogenic activity (ER-␣ agonist). Immunomodulating effects as induced by estrogens are pleiotropic and may lead to both enhancement and suppression of many aspects of the immune response. Sex steroid hormones are important factors that contribute to the noted strong sexual dimorphism in the mammalian immune response. Males generally exhibit lower immune responses than females (Cernetich et al., 2006; Klein, 2000; Schuurs and Verheul, 1990). Conversely, females from many species have been shown to be more prone to producing immune responses against self-tissues
∗ Corresponding author. Tel.: +31 412 666192; fax: +31 412 666131. E-mail address: [email protected] (G.B. Janssen). 0378-4274/$ – see front matter © 2008 Elsevier Ireland Ltd. All rights reserved. doi:10.1016/j.toxlet.2008.06.857
and thus exhibit an increased tendency to develop autoimmune diseases in comparison to male individuals. In addition, an elevated humoral immune response (i.e. antibody production by B-cells) has been demonstrated in females. In male rats physiological levels of estrogen enhances immune response by increasing the synthesis of IgM antibodies (Myers and Petersen, 1985). The cell-mediated immune response also differs between males and females with females exhibiting a more pronounced Th2 response than males (Myers and Petersen, 1985). Although physiological levels of estrogens are immunostimulatory, supraphysiological levels appear to be immunosuppressive (Kovacs et al., 2002). Levels of estrogen during pregnancy may be 100–1000 times that of cycling females. This suppresses maternal Th1 responses in order to protect the conceptus and it attenuates the severity of certain disorders which are mediated by aberrant immune function, such as autoimmune diseases (Kovacs et al., 2002). Likewise, estrogen treatment protects female mice from experimental autoimmune encephalomyelitis by inhibiting the recruitment of T cells and macrophages into the CNS (Subramanian et al., 2003). In pregnant mice B cell lymphopoiesis appeared to
G.B. Janssen et al. / Toxicology Letters 180 (2008) 196–201
197
in the rat (Andrews et al., 2002b). Moreover, in previous in-house performed sub-acute toxicity experiments (data not shown) a dose level of 0.05 mg/kg EE (pharmacological dose) produced a slightly less decreased body weight in female Sprague–Dawley rats than a dose of 0.2 mg/kg ERA-63 in Wistar rats. Hence these dosages and 10 times multiples were selected for the present studies. Besides a qualitative comparison (hazard identification) of immunomodulatory effects the parallel testing with EE and the evaluation at estrogenic equipotent dose levels allowed also for a quantitative risk assessment in case similar results would be obtained. 2. Materials and methods 2.1. Test chemical
Fig. 1. Structures of ERA-63 and EE.
be selectively suppressed due to the increased levels of estrogen (Medina and Kincade, 1994). In rats estrogens enhance antibody production in the physiological range and suppress antibody production in the pharmacological range (Trawick and Bahr, 1986). Female mice treated with either physiological or pharmacological doses of estrogens are more susceptible to Listeria monocytogenes, Salmonella typhimurium and Toxoplasma gondii than non-treated female or males (Kita et al., 1985, 1989; Pung et al., 1985). Functional estrogen receptor expression is required for both the humoral (Erlandsson et al., 2003; Smithson et al., 1998; Thurmond et al., 2000) and the cell-mediated immune response (Huber et al., 1999; Liu et al., 2003; Maret et al., 2003). The two nuclear estrogen receptor isoforms (ER␣ and ERß) are differentially expressed according to immune cell type (Pillet et al., 2006). Mechanisms by which estrogen affect the functionality of the immune system may be (i) regulation of cytokine expression, (ii) affecting the elimination of autoreactive cells (negative and positive selection) in development organs (e.g. thymus and bone marrow) through an effect on the morphology and (iii) altering the patterns of apoptosis of T and B cells (Ahmed, 2000). In summary, the above indicates that interactions of estrogenic compounds are complex and due to the induced pleiotropic effects not easily predictable. The overall aim of the performed studies (the PFC assay and the host resistance model using L. monocytogenes) was to investigate whether ERA-63, a compound with intended immunomodulatory effects and having estrogenic activity, showed immunomodulating properties that could not be attributed to it’s estrogenic activity. Both the PFC assay (testing the T cell-dependent antibody response to SRBC) and the host resistance assay (testing the non-specific immunity towards a L. monocytogenes infection) are mentioned in the ICH S8 guideline for immunotoxicity testing of human pharmaceuticals as recommended functional immune tests to screen for potential immunotoxicity of compounds, and are expected to give sufficient data for risk assessment and risk management. Effects of test compounds in the PFC assay alone are considered to be about 80% predictive for immunotoxicity (Luster et al., 1992). Estrogenic potency was assessed mainly by the indirect parameter body weight as in toxicological experiments this parameter appeared to be a very sensitive parameter for estrogenic activity
Ethinyl estradiol (19-nor-17alpha-pregna-1,3,5[10]-trien-20-yne-3, 17beta-diol, CAS no 57-63-6, purity 100%) was originally obtained from N.V. Organon, Oss, The Netherlands. It was formulated by the Investigational Products Supply Department, Organon S.A., Riom, France, as a freeze dried preparation. After reconstitution with water for injection a suspension containing 5 mg ethinyl estradiol/mL in gelatin/mannitol 0.5%/5% (w/v) was obtained. Final dosing preparations were prepared by dilution with gelatin/mannitol 0.5%/5% (w/v). ERA-63 ((7-alpha,17-alpha)-7-methyl-3-methylene-19-norpregn-5[10]-en-20yn-17-ol, solution in ethanol) was originally obtained from N.V. Organon, Oss, The Netherlands. It was formulated by the Investigational Products Supply Department, Organon S.A., Riom, France, as a suspension containing 50 mg ERA-63/mL, gelatin/mannitol 0.5%/5% (w/v) and ethanol <2%. Final dosing preparations were prepared by dilution with gelatin/mannitol 0.5%/5% (w/v). Cyclophosphamide, i.v. lyophilisate (CPS, Product code: RVG 08058) was obtained from Asta Medica, Frankfurt/Main, Germany. A solution of 25 mg/mL (males) or 12.5 mg/mL (females) was prepared in water for injection shortly before administration. 2.2. Animals Male and female rats HSD/CpB: WU (spf-bred) obtained from Charles River Deutschland, Sulzfeld, Germany, were kept in macrolon cages with a soft wood bedding (four animals of one sex per cage, except for the control group of the plaqueforming cell assay in which five animals of the same sex were housed per cage). The rats were fed ad libitum using a standard pelleted rodent diet (RM3 (E) SQC, obtained from SDS Special Diets Services, Whitham, England). Animals were 6–8 weeks old upon arrival and 8–10 weeks at the start of dosing. Body weight range at the start of dosing was 158–201 g for females and 219–287 g for males in the plaque-forming cell assay and 135–188 g for females and 184–253 g for males in the host resistance assay. The animal room had a 12-h light/dark regime, a temperature of 21 ± 2 ◦ C, a relative humidity of 30–71%, and a rate of air exchange of approximately 10 times per hour. All procedures used in this experiment were compliant with the regional ethics regulations and approved by a local ethics committee. 2.3. Experimental design Two studies were performed each consisting of eight groups of male and female rats dosed orally with the vehicle (control group), ethinyl estradiol at a low dose of 0.05 mg/kg, a mid dose of 0.5 mg/kg and a high dose of 5 mg/kg (comprising a dose range starting at approximately the pharmacological dose up to clearly toxic levels), ERA-63 at a low dose of 0.2 mg/kg, a mid dose of 2 mg/kg and a high dose of 20 mg/kg (comprising a dose range starting at approximately the pharmacological dose up to clearly toxic levels), and cyclophosphamide at a dose level of 50 mg/kg (males in the host resistance assay received 100 mg cyclophosphamide/kg). Due to human error the dose levels ERA-63 in “Study II: host resistance assay with Listeria monocytogenes” were from the second day of dosing onwards 0.167, 1.67 and 16.7 mg/kg for the low, mid and high dose, respectively, instead of the above-mentioned dosages. The control group, the ethinyl estradiol treated groups and the ERA-63 treated groups were treated daily by gavage (1 mL/kg) for 29 days (first day of treatment is day 0). Cyclophosphamide was administered once intravenously (4 mL/kg) on day 27 in the host resistance assay and on day 24 in the plaque-forming cell assay (on both occasions the day before challenge with antigen or pathogen). 2.3.1. Study I: T cell-dependent antibody response in the plaque-forming cell (PFC) assay In the plaque-forming cell (PFC) assay eight animals/(group sex) (ten animals/sex for the control group) were dosed as indicated above. On day 24, all groups received an i.v. injection of 0.5 mL with 5 ± 1 × 108 sheep red blood cells (SRBC, Biotrading Benelux b.v., Mijdrecht, The Netherlands). Autopsy was performed on day 29. The animals were sacrificed using CO2 /O2 and spleen was collected aseptically, weighed and further processed to determine the number of plaque-forming cells, in addition, the thymus was weighed.
198
G.B. Janssen et al. / Toxicology Letters 180 (2008) 196–201
2.3.2. Study II: host resistance assay with L. monocytogenes In the host resistance assay sixteen animals/(group sex) were dosed as indicated above. All groups were divided over two subgroups (S1 and S2) each containing eight animals. All animals were infected (i.v.) with approximately 5 × 105 (as determined by means of fluorescent staining and flow cytometric counting and subsequently confirmed by plating and determination of the number CFU) L. monocytogenes bacteria (type 4b, source: National Institute of Health and Environmental Hygiene (RIVM)) in 500 l phosphate-buffered salt solution on day 28 (after the last dosing with test item). To provide the L. monocytogenes bacteria with sufficient virulence for experimental infections in the rat, the bacteria had been activated, prior to the administration, by culturing on fertilized and 10-day hard set eggs. Two and four days after infection the animals of subgroups S1 and S2, respectively, were anesthetized and killed by exsanguination from the abdominal aorta. Liver and spleen were removed aseptically, collected in sterile petri dishes, weighed and cooled on ice. The liver and spleen samples were transferred to the microbiological laboratory, and further processed for the quantitative detection of viable L. monocytogenes bacteria (see below). The culture of liver and spleen samples started on the day of removal of the organs. In addition, thymus, ovaries, and testis were removed and weighed. 2.4. Clinical observations and body weights The general condition and behavior of all animals was checked and recorded at least once daily. Body weights were determined twice weekly, on the day of CPS injection (positive control group), challenge (PFC assay), infection (host resistance assay) and at necropsy (all animals). 2.5. Clinical pathology Clinical pathology was conducted on the animals used for the in the host resistance assay. On day 25 blood samples were collected from the orbital plexus of all animals and on day 30 (day 2 post-infection) and 32 (day 4 post-infection) blood samples were collected at necropsy from the abdominal aorta from the two subgroups. K2 -EDTA was used as anticoagulant for hematology and blood clotting and lithium–heparin was used as anticoagulant for clinical biochemical parameters. The following hematological parameters were determined: hemoglobin, packed cell volume, red blood cell count, reticulocytes, total white blood cell count, differential white blood cell count, prothrombin time (PTT), thrombocyte count, mean corpuscular volume, mean corpuscular hemoglobin and mean corpuscular hemoglobin concentration. The following clinical biochemical parameters were determined: alkaline phosphatase activity (ALP), aspartate aminotransferase activity (ASAT) alanine aminotransferase activity (ALAT), ␥-glutamyl transferase activity (GGT), bilirubin (total), cholesterol (total), triglycerides, phospholipids, total protein, albumin, ratio albumin to globulin, urea, creatinine, sodium, potassium, calcium, chloride and inorganic phosphate. 2.6. Microbiological determination of L. monocytogenes in liver and spleen Liver and spleen samples were homogenized in sterile physiological salt solution (PSS). A tenfold amount of sterile PSS was added aseptically to each of the samples. These samples were then homogenized on ice during 60 s. Further decimal dilutions (10−2 , 10−3 and 10−4 ) were prepared in PSS and 0.1 mL was plated on non-selective sheep-blood-agar plates (Oxoid, Haarlem, the Netherlands) in duplicate. After addition of glycerol (final concentration 10%), the remaining homogenates were stored at −80 ◦ C. The plates were incubated under aerobic conditions for 48 h at 37 ◦ C and subsequently 24 h at room temperature. Typical L. monocytogenes colonies developed on the sheep-blood-agar plates were counted, and the number of colony forming units (CFU) per gram or total liver or spleen was calculated. Ten bacterial strains isolated from randomly selected colonies were used for L. monocytogenes conformational testing using the Microbact L12 system. 2.7. Determination of SRBC-specific plaque-forming cells in spleen cell suspensions The spleens were collected in a vial containing 5 mL RPMI medium. Single cell suspensions were prepared by gently rubbing the spleens through a 200 m steel filter by means of a blunt object. After removing the cell suspension, rinsing took place with 5 mL of RPMI, which was also collected. The numbers of spleen cells in RPMI medium were determined using an automated haematology analyser (K-800, Sysmex Toa, Kobe, Japan). Viability was assessed using an acridine orange/ethidium bromide labelling procedure. A suspension with a final viable spleen cell concentration of 10E+06 cells/mL was prepared. The plaque-forming cell (PFC) assay was performed according to the method described by Cunningham and Szenberg (1968). In short, three dilutions of the spleen cell suspensions (1, 0.5 and 0.25 × 10E+06 vital spleen cells) were tested in duplicate. The spleen cell suspensions were incubated with SRBC in the presence of (guinea pig) complement during 90 min at 37 ◦ C. Using a low magnification the formed plaques were counted and expressed as number of PFC/10E+06 spleen
cells and as number of PFC/spleen. If feasible, calculations were based on spleen cell dilutions with PFC counts between 50 and 100 PFC counted per chamber, since these counts are considered most reliable. 2.8. Statistical analysis of results The survival of L. monocytogenes as reflected by the number of CFU obtained from (liver or spleen) samples of the test subgroups were compared with the corresponding control subgroups by using analysis of (co)variance followed by Dunnet’s multiple comparison tests. CFU data were log transformed to obtain normal distributions. Differences in body and organ weights, hematological findings, total and differential white blood cell counts and clinical chemistry were analysed by analysis of (co)variance followed by Dunnet’s multiple comparison tests or by one-way analysis of variance followed by Dunnett’s multiple comparison tests. In case of inhomogeneous variances (tested by means of Bartlett’s test), or in case of non-continuous parameters: Kruskal–Wallis non-parametric analysis of variance followed by Mann–Whitney U-tests was performed. PFC data were evaluated by means of Brown–Forsythe ANOVA.
3. Results 3.1. General toxicity Most relevant changes in general toxicity parameters observed in both the T cell-dependent antibody response (plaque-forming cell (PFC) assay) and the host resistance assay with L. monocytogenes are summarized in Table 1. Males and females treated with EE or ERA-63 showed statistically significant dose-dependent decreases in body weight from day 3 onward in all dose groups, which resulted in a dose-dependent decrease in the terminal body weights (see Fig. 2) and a generally thin appearance at the mid- and high dose EE and ERA-63 treated groups. Additionally, the males at these dose levels showed severely reduced testis size. As expected EE and ERA-63 treatment also affected the hematological system, liver function, and the weights of spleen, thymus and testis. Regarding hematological changes relevant and statistically significant decreases in red blood cell count, hemoglobin concentration, packed cell volume and increased reticulocyte count were noted (data not shown). White blood cell and lymphocyte counts were decreased in the high dose groups with both compounds (Table 1) in both sexes. Indicative for changes in liver function were a dose-related increase in GGT, total bilirubin and prothrombin time (Table 1) and a dose-related decrease in albumin/globulin ratio. With a few exceptions, these changes were in all dose groups statistically significant. Lipid metabolism was also (statistically significant with a few exceptions) affected as indicated by decreased cholesterol and phospholipids at all dose groups and increased triglycerides in the low- and mid dose groups and decreased triglycerides in the high dose groups (data not shown) with both compounds. Organ weights such as, male testis, and for both sexes thymus and spleen weights were dose dependently and statistically significantly decreased in the EE and the ERA-63 treated groups of both sexes (Table 1). 3.2. T cell-dependent antibody response as measured by plaque-forming cell (PFC) assay Results of the relevant parameters of the PFC assay are summarized in Table 2 and the number of PFC per spleen is depicted in Fig. 3. In line with the decreased spleen weight, the number of viable cells per spleen was dose dependently decreased in both sexes (except the low dose females) with both test compounds. Regarding the numbers of PFC cells per spleen especially in males statistically significant decreases of around 50% were noted for both EE and ERA-63 at all dose levels tested. Females showed less pronounced
G.B. Janssen et al. / Toxicology Letters 180 (2008) 196–201
199
Table 1 General toxicity parameters in male and female Wistar rats after treatment for 29 days with 0 mg/kg (control), 0.05 mg/kg, 0.5 mg/kg or 5 mg/kg ethinyl estradiol (EE), or 0.167 mg/kg, 1.67 mg/kg or 16.7 mg/kg ERA-63 (mean results and standard deviation) Parameter
Control
Ethinyl estradiol (mg/kg)
0 mg/kg
0.05
ERA-63 (mg/kg)
0.5
5
0.167
1.67
16.7
Males BW day 28 (g) BW day 28 (g) P PTT (s) WBC (109 cells/L) Lymphocytes (109 cells/L) GGT (U/L) Bilirubin (mol/L) Thymus (g) P Spleen (g) P Testis (g) P
332.6 330.6 41.6 14.1 12.8 0.1 0.9 0.588 0.823 3.35
± ± ± ± ± ± ± ± ± ±
5.2 6.8 0.4 0.4 0.4 0.1 0.1 0.023 0.025 0.06
270.6 267.1 44.9 15.6 14.5 0.1 2.4 0.407 0.700 2.88
± ± ± ± ± ± ± ± ± ±
4.6** 6.4** 0.7$$$ 0.8 0.8 0.0 0.2$$$ 0.023$$$ 0.019* 0.12**
216.3 212.8 48.7 14.3 13.3 0.8 4.1 .270 0.554 0.62
± ± ± ± ± ± ± ± ± ±
4.4** 6.2** 0.4$$$ 0.5 0.5 0.1$$$ 0.3$$$ 0.032$$$ 0.033** 0.02**
177.1 194.2 50.9 12.6 11.5 5.6 7.8 0.171 0.565 0.57
± ± ± ± ± ± ± ± ± ±
5.4** 9.8** 1.1$$$ 0.6 0.5 0.5$$$ 0.5$$$ 0.016$$$ 0.018** 0.02**
236.2 230.8 48.5 15.7 14.8 0.1 2.7 0.345 0.549 2.45
± ± ± ± ± ± ± ± ± ±
4.4** 2.1** 0.7$$$ 0.6 0.6 0.0 0.2$$$ 0.020 0.015** 0.11**
208.4 201.8 49.5 14.2 13.0 0.5 4.1 0.229 0.516 0.59
± ± ± ± ± ± ± ± ± ±
5.3** 6.2** 0.9$$$ 0.6 0.5 0.1$$ 0.3$$$ 0.030$$$ 0.024** 0.03**
177.0 181.4 48.5 11.7 10.5 2.3 6.6 0.129 0.463 0.74
± ± ± ± ± ± ± ± ± ±
4.6** 5.5** 0.7$$$ 0.6* 0.6* 0.3$$$ 0.6$$$ 0.006$$$ 0.051** 0.11**
Females BW day 28 (g) BW day 28 (g) P PTT (s) WBC (109 cells/L) Lymphocytes (109 cells/L) GGT (U/L) Bilirubin (mol/L) Thymus (g) P Spleen (g) P
200.4 211.5 40.0 11.1 10.5 1.1 0.8 0.390 0.563
± ± ± ± ± ± ± ± ±
4.1 13.6 0.9 0.6 0.5 0.1 0.4 0.022 0.016
181.1 193.8 46.6 13.1 12.2 1.4 1.5 0.385 0.556
± ± ± ± ± ± ± ± ±
2.6** 8.7** 0.7** 0.7 0.7 0.1 0.4$$ 0.015 0.012
151.9 158.4 48.7 11.8 10.9 2.6 2.6 0.224 0.517
± ± ± ± ± ± ± ± ±
2.8** 8.6** 1.0** 0.7 0.6 0.2$$$ 0.3$$$ 0.014** 0.024
129.1 147.9 51.5 8.7 7.3 5.5 6.3 0.132 0.475
± ± ± ± ± ± ± ± ±
2.1** 6.5** 1.2** 0.7* 0.6** 0.6$$$ 0.7$$$ 0.011** 0.012**
185.3 196.2 41.8 12.8 11.8 1.3 0.4 0.427 0.583
± ± ± ± ± ± ± ± ±
2.3** 3.6* 0.9 0.6 0.6 0.1 0.1 0.037 0.028
150.3 171.2 48.7 12.7 11.5 1.3 4.1 0.246 0.429
± ± ± ± ± ± ± ± ±
3.2** 4.6** 1.0** 0.8 0.7 0.1 0.8$$$ 0.017** 0.015*
133.8 141.1 52.7 8.0 6.7 4.5 5.4 0.104 0.425
± ± ± ± ± ± ± ± ±
2.8** 3.4** 1.1** 0.6** 0.5** 0.5$$$ 0.6$$$ 0.009** 0.014**
Statistics homogenous data: one-way analysis of variance followed by Dunnett’s multiple comparison test (*, p < 0.05; **, p < 0.01), inhomogeneous data: Kruskal–Wallis non-parametric analysis of variance followed by Mann–Whitney U-test ($, p < 0.05; $$, p < 0.02; $$$, p < 0.002). P = determined in plaque-forming cell assay with dosages ERA-63 of 0.2, 2.0 and 20 mg/kg instead of mentioned dosages.
Fig. 3. Mean number of plaque-forming cells (PFC) per spleen in the PFC assay after treatment for 29 days with 0 mg/kg (control), 0.05 mg/kg, 0.5 mg/kg or 5 mg/kg ethinyl estradiol (EE) or 0 mg/kg (control), 0.167 mg/kg, 1.67 mg/kg or 16.7 mg/kg ERA-63 in male and female Wistar rats.
Fig. 2. Mean body weights after treatment for 29 days with 0 mg/kg (control), 0.05 mg/kg, 0.5 mg/kg or 5 mg/kg ethinyl estradiol (EE) or 0 mg/kg (control), 0.167 mg/kg, 1.67 mg/kg or 16.7 mg/kg ERA-63 in male and female Wistar rats.
Table 2 Plaque-forming cell (PFC) parameters in male and female Wistar rats after treatment for 29 days with 0 mg/kg (control), 0.05 mg/kg, 0.5 mg/kg or 5 mg/kg ethinyl estradiol (EE), or 0.2 mg/kg, 2.0 mg/kg or 20 mg/kg ERA-63 (mean results and standard deviation) Parameter
Values
Ethinyl estradiol (mg/kg)
0 mg/kg
0.05
ERA-63 (mg/kg)
0.5
5
0.2
2.0
20
Males Viability (%) Viable cells/spleen (×108 ) PFC per 106 cells PFC per spleen (×108 )
66.8 4.2 8079 3363
± ± ± ±
5.3 1.2 4099 1771
61.9 3.2 5257 1528
± ± ± ±
4.8 0.8 3030 762*
61.0 2.2 7739 1834
± ± ± ±
3.1 0.5** 5271 1607
58.6 2.0 10,319 1947
± ± ± ±
3.5 0.6** 4872 830
68.6 3.4 5413 1741
± ± ± ±
3.2 0.8 2803 1021*
67.0 2.6 6439 1669
± ± ± ±
5.0 0.6** 2549 713*
65.1 2.3 8450 2025
± ± ± ±
6.3 0.5** 4276 1214
Females Viability (%) Viable cells/spleen (×108 ) PFC per E106 cells PFC per spleen (×108 )
67.1 3.4 8717 2918
± ± ± ±
6.8 0.8 3530 1362
69.3 3.4 8560 2956
± ± ± ±
3.2 0.6 4793 1750
65.1 2.9 5688 1713
± ± ± ±
2.8 0.7 2458 947
63.6 2.1 10,844 2216
± ± ± ±
3.7 0.4** 5281 1061
64.3 3.2 9464 2746
± ± ± ±
6.6 1.0 3484 980
58.3 2.2 9811 2144
± ± ± ±
4.2 0.4* 2298 679
56.8 1.8 11,435 1989
± ± ± ±
4.2 0.4** 3187 492
Statistics homogenous data: one-way analysis of variance followed by Dunnett’s multiple comparison test (*, p < 0.05; **, p < 0.01), inhomogeneous data: Brown-Forsythe one-way analysis of variance (*, p < 0.05; ** p < 0.01).
200
G.B. Janssen et al. / Toxicology Letters 180 (2008) 196–201
treated with ERA-63 the number of CFU per organ was decreased 4 days post-infection as compared to the mid dose ERA-63 group and the high dose EE group, however this effect may be biased because the values of the high dose group was based on only four surviving animals. In spleen the pathogen burden for control animals and animals treated with low doses of EE and ERA-63 was 4 days post-infection lower or comparable to the pathogen burden 2 days post-infection. Especially in males treated with the higher doses of EE or ERA63 dose-related increases in the numbers of CFU per spleen were noted, which were statistically significant at the highest dose. In female animals these increases were much less pronounced and not statistically significant. In the high dose ERA-63 treated females the number of CFU per spleen was decreased 4 days postinfection as compared to the mid dose ERA-63 and the high dose EE groups. As remarked for the liver findings this effect may be biased because the value of the high dose group was based on only four surviving animals. The positive control group treated with CPS (data not shown) showed mortality on day 31 (4/8 males) and day 32 (2/8 females). The remaining animals that were sacrificed showed statistically significant increased numbers of viable L. monocytogenes bacteria recovered from liver and spleen. Four days after infection the number of viable L. monocytogenes bacteria recovered from liver and spleen was increased as compared to day 2 of infection. These results signify the validity of the infection model. Fig. 4. Mean number of colony forming units (CFU) per total liver and spleen in the host resistance assay with Listeria monocytogenes after treatment for 29 days with 0 mg/kg (control), 0.05 mg/kg, 0.5 mg/kg or 5 mg/kg ethinyl estradiol (EE) or 0 mg/kg (control), 0.2 mg/kg, 2 mg/kg or 20 mg/kg ERA-63 in male and female Wistar rats.
reductions at the mid and the high dose levels as is reflected by the magnitude of effects (around 30%, not statistically significant). Both for males and females a plateau effects was reached already from the low- and mid dose, respectively. Due to the overall decreases in spleen cell numbers, the number of PFC per 106 spleen cells tended to go up at the high dose levels tested. Animals treated with the positive control compound, CPS, showed as expected statistically significantly decreased thymus and spleen weights and spleen cell numbers. The number of PFC per spleen or per 106 spleen cells was negligible, indicating that CPS as positive control already demonstrated the functionality of the test system. 3.3. Host resistance assay with L. monocytogenes The number of colony forming units (CFU) per total spleen and liver, as determined in the host resistance assay with L. monocytogenes are depicted in Fig. 4a and b, respectively. Two days after infection (i.e. day 30) 1/16 females of the mid dose group EE as well as 4/16 males and 4/16 females of the high dose group ERA-63 died. From the remaining animals 1/7 males died of the high dose group ERA-63, 3 days post-infection and 4 days post-infection 1/8 males of the mid dose group ERA-63 and 1/5 females of the high dose ERA-63 died. In general, with the exception of the control group for males, the pathogen burden in the liver was higher 4 days post-infection as compared to 2 days post-infection, independent of treatment. Both EE and ERA-63 treatment especially at the higher dose levels showed dose-related increased numbers of CFU per organ 2 and 4 days post-infection. Apart from a difference in the high dose females at day 4 post-infection, the effects of ERA-63 as compared to EE showed no large quantitative differences in the numbers of CFU’s invoked by either the EE or ERA-63 treatment. In females
4. Discussion In order to facilitate a proper risk assessment of ERA-63 the overall aim of the performed studies was to investigate whether ERA-63, a compound with estrogenic activity in rats, showed additional immunomodulatory effects besides those due to its estrogenic activity. As will be discussed below, a comparison with EE at estrogenic equipotent dose levels showed neither qualitative nor quantitative differences between immunomodulating effects of the compounds. This indicates that for human risk assessment it is expected that ERA-63 treatment would not pose a different risk on an immunotoxic insult as compared to EE at equipotent dose levels. A number of parameters showed clear dose-related effects for both EE and ERA-63 and were considered to reflect estrogenic activity in rats based on literature data. These parameters were: body weight, thymus and testis organ weight, bilirubin and GGT levels. Changes in body weight are a direct consequence of an estrogenic effect on overall metabolism (Dagnault and Richard, 1997). Reduced thymus weight is related to estrogen-induced thymic atrophy (Yao and Hou, 2004) and reduced testis weight is in accordance with the known activity of estrogens acting as functional antiandrogens (antagonistic for androgens) in the male rat (Andrews et al., 2002a). Another well-known effect of EE in rats is induction of cholestasis (Azer et al., 1995), which is reflected amongst others by increased bilirubin and GGT levels. In the host resistance study the results of a number of the abovementioned parameters might have been confounded by the (degree of) infection. Therefore, the clinical pathology parameters determined pre-infection in the host resistance assay and the organ weights obtained from the PFC assay were used to compare the estrogenic effects of ERA-63 and EE. Calculation of dose–response curves for the parameters reflecting estrogenic activity allowed determination of the estrogenic potency of ERA-63 as compared to EE. As expected, based on the effects observed, the estrogenic potencies for the different dose groups treated with ERA-63 and EE were more or less comparable. However, there was some variability
G.B. Janssen et al. / Toxicology Letters 180 (2008) 196–201
depending the parameter investigated and the dose level considered. For some parameters (e.g. bilirubine and GGT) the estrogenic potency at the high dose ERA-63 was lower than the high dose EE. However, for the stronger estrogen-related parameters, e.g. body weight, growth, and thymus weight, the estrogenic potency of the high dose ERA-63 was around two times higher than the high dose EE. When considering results of the PFC both compounds showed more or less similar effects. Regarding the host resistance assays it appeared that effects of ERA-63 were, although similar, more pronounced as reflected by a higher mortality at the high dose level. However, this can be explained by the calculated higher potency of ERA-63 as compared to EE at the high dose level for some parameters. Overall, based on the similarities between noted effects of both compounds, the results demonstrated that apart from a quantitative difference there is no qualitative difference between ERA-63 and EE. Thus with respect to risk assessment this compound may be compared to EE with regard to anticipated effects on the human immune system. Although more pronounced in males, both compounds reduce the T cell-dependent antibody response in both sexes. The reduced IgM response against SRBC in the suprapharmacological range of EE is in line with findings described by others (Myers and Petersen, 1985). The dose-dependent increases in the number of viable L. monocytogenes bacteria recovered from spleen and from liver (per gram and per organ) both in the ERA-63 and EE treated groups indicate an increased sensitivity to infection. This finding is in line with similar findings in mice treated with estrogenic compounds (Pung et al., 1985). It is concluded that ERA-63 show immunomodulatory effects comparable to those of EE, and as earlier described for estrogens, i.e. decreases in specific antibody responses and an increased susceptibility for L. monocytogenes infects. Comparison of ERA-63 with EE at equipotent dose levels showed neither qualitative nor quantitative differences between the compounds. This indicates that for human risk assessment it is expected that ERA-63 administered to humans would not pose a different risk on an immunotoxic insult as compared to EE. Any anticipated effect in humans is likely to be similar to what is known with respect to equipotent EE levels. Conflict of interest None. References Ahmed, S.A., 2000. The immune system as a potential target for environmental estrogens (endocrine disrupters): a new emerging field. Toxicology 150, 191–206. Andrews, P., Freyberger, A., Hartmann, E., Eiben, R., Loof, I., Schmidt, U., Temerowski, M., Folkerts, A., Stahl, B., Kayser, M., 2002 a. Sensitive detection of the endocrine effects of the estrogen analogue ethinylestradiol using a modified enhanced subacute rat study protocol (OECD Test Guideline no. 407). Arch. Toxicol. 76, 194–202. Andrews, P., Freyberger, A., Hartmann, E., Eiben, R., Loof, I., Schmidt, U., Temerowski, M., Folkerts, A., Stahl, B., Kayser, M., 2002 b. Sensitive detection of the endocrine
201
effects of the estrogen analogue ethinylestradiol using a modified enhanced subacute rat study protocol (OECD Test Guideline no. 407). Arch. Toxicol. 76, 194–202. Azer, S.A., Canfield, P.J., Stacey, N.H., 1995. Hepatoprotection in ethinylestradioltreated rats is provided by tauroursodeoxycholic acid, but not by ursodeoxycholic acid. J. Gastroenterol. Hepatol. 10, 261–269. Cernetich, A., Garver, L.S., Jedlicka, A.E., Klein, P.W., Kumar, N., Scott, A.L., Klein, S.L., 2006. Involvement of gonadal steroids and gamma interferon in sex differences in response to blood-stage malaria infection. Infect. Immun. 74, 3190–3203. Cunningham, A.J., Szenberg, A., 1968. Further improvements in the plaque technique for detecting single antibody-forming cells. Immunology 14, 599–600. Dagnault, A., Richard, D., 1997. Involvement of the medial preoptic area in the anorectic action of estrogens. Am. J. Physiol. 272, R311–R317. Dulos, J., Hofstra, C.L., Joosten, L.A.B., Veening-Griffioen, D.H., Lucassen, M.A., Doorn, C.M., Graaf, J.H., Miltenburg, A.M.M., Dijcks, F.A., Ederveen, T.H., Boots, A.M.H., 2006. A selective estrogen receptor alpha agonist (Org 37663) suppresses inflammation and arthritis in mouse models. Ann. Rheum. Dis. 65 (suppl. II), 128. Erlandsson, M.C., Jonsson, C.A., Islander, U., Ohlsson, C., Carlsten, H., 2003. Oestrogen receptor specificity in oestradiol-mediated effects on B lymphopoiesis and immunoglobulin production in male mice. Immunology 108, 346–351. Huber, S.A., Kupperman, J., Newell, M.K., 1999. Estradiol prevents and testosterone promotes Fas-dependent apoptosis in CD4+ Th2 cells by altering Bcl 2 expression. Lupus 8, 384–387. Kita, E., Takahashi, S., Yasui, K., Kashiba, S., 1985. Effect of estrogen (17 betaestradiol) on the susceptibility of mice to disseminated gonococcal infection. Infect. Immun. 49, 238–243. Kita, E., Yagyu, Y., Nishikawa, F., Hamuro, A., Oku, D., Emoto, M., Katsui, N., Kashiba, S., 1989. Alterations of host resistance to mouse typhoid infection by sex hormones. J. Leukoc. Biol. 46, 538–546. Klein, S.L., 2000. The effects of hormones on sex differences in infection: from genes to behavior. Neurosci. Biobehav. Rev. 24, 627–638. Kovacs, E.J., Messingham, K.A., Gregory, M.S., 2002. Estrogen regulation of immune responses after injury. Mol. Cell. Endocrinol. 193, 129–135. Liu, H.B., Loo, K.K., Palaszynski, K., Ashouri, J., Lubahn, D.B., Voskuhl, R.R., 2003. Estrogen receptor alpha mediates estrogen’s immune protection in autoimmune disease. J. Immunol. 171, 6936–6940. Luster, M.I., Portier, C., Pait, D.G., White Jr., K.L., Gennings, C., Munson, A.E., Rosenthal, G.J., 1992. Risk assessment in immunotoxicology. I. Sensitivity and predictability of immune tests. Fundam. Appl. Toxicol. 18, 200–210. Maret, A., Coudert, J.D., Garidou, L., Foucras, G., Gourdy, P., Krust, A., Dupont, S., Chambon, P., Druet, P., Bayard, F., Guery, J.C., 2003. Estradiol enhances primary antigen-specific CD4 T cell responses and Th1 development in vivo. Essential role of estrogen receptor alpha expression in hematopoietic cells. Eur. J. Immunol. 33, 512–521. Medina, K.L., Kincade, P.W., 1994. Pregnancy-related steroids are potential negative regulators of B lymphopoiesis. Proc. Natl. Acad. Sci. U. S. A. 91, 5382–5386. Myers, M.J., Petersen, B.H., 1985. Estradiol induced alterations of the immune system. I. Enhancement of IgM production. Int. J. Immunopharmacol. 7, 207–213. Pillet, S., D’Elia, M., Bernier, J., Bouquegneau, J.M., Fournier, M., Cyr, D.G., 2006. Immunomodulatory effects of estradiol and cadmium in adult female rats. Toxicol. Sci. 92, 423–432. Pung, O.J., Tucker, A.N., Vore, S.J., Luster, M.I., 1985. Influence of estrogen on host resistance: increased susceptibility of mice to Listeria monocytogenes correlates with depressed production of interleukin 2. Infect. Immun. 50, 91–96. Schuurs, A.H., Verheul, H.A., 1990. Effects of gender and sex steroids on the immune response. J. Steroid Biochem. 35, 157–172. Smithson, G., Couse, J.F., Lubahn, D.B., Korach, K.S., Kincade, P.W., 1998. The role of estrogen receptors and androgen receptors in sex steroid regulation of B lymphopoiesis. J. Immunol. 161, 27–34. Subramanian, S., Matejuk, A., Zamora, A., Vandenbark, A.A., Offner, H., 2003. Oral feeding with ethinyl estradiol suppresses and treats experimental autoimmune encephalomyelitis in SJL mice and inhibits the recruitment of inflammatory cells into the central nervous system. J. Immunol. 170, 1548–1555. Thurmond, T.S., Murante, F.G., Staples, J.E., Silverstone, A.E., Korach, K.S., Gasiewicz, T.A., 2000. Role of estrogen receptor alpha in hematopoietic stem cell development and B lymphocyte maturation in the male mouse. Endocrinology 141, 2309–2318. Trawick, D.R., Bahr, J.M., 1986. Modulation of the primary and secondary antifluoresceyl antibody response in rats by 17 beta-estradiol. Endocrinology 118, 2324–2330. Yao, G., Hou, Y., 2004. Thymic atrophy via estrogen-induced apoptosis is related to Fas/FasL pathway. Int. Immunopharmacol. 4, 213–221.