Use of high-dose nandrolone aggravates septic shock in a mouse model

Kaohsiung Journal of Medical Sciences (2011) 27, 222e229

available at www.sciencedirect.com

journal homepage: http://www.kjms-online.com

ORIGINAL ARTICLE

Use of high-dose nandrolone aggravates septic shock in a mouse model 高劑量同化性類固醇Nandrolone對敗血性休克的影響d小鼠模式研究 Che Lin a,b,c,1, Shou-Tung Chen a,1, Su-Yu Chien d,e, Shou-Jen Kuo a,b,e,f, Dar-Ren Chen a,b,c,f,* 林喆

a,b,c,1

, 陳守棟

a,1

, 簡素玉

, 郭守仁

d,e

, 陳達人

a,b,e,f

a,b,c,f,

*

a

Division of General Surgery, Department of Surgery, Changhua Christian Hospital, Changhua, Taiwan Comprehensive Breast Cancer Center, Changhua Christian Hospital, Changhua, Taiwan c Department of Medical Research, Changhua Christian Hospital, Changhua, Taiwan d Department of Pharmacology, Changhua Christian Hospital, Changhua, Taiwan e College of Health Care and Management, Chung Shan Medical University, Taichung, Taiwan f School of Medicine, Chung Shan Medical University, Taichung, Taiwan b

Received 26 August 2010; accepted 7 December 2010 Available online 31 March 2011

KEYWORDS Anabolic-androgenic steroid; Insulin-like growth factor; Nandrolone; Septic shock

關鍵詞

同化性類固醇; 類胰島素生長因子; 諾龍; 敗血性休克

Abstract Nandrolone, an anabolic-androgenic steroid, is widely misused by athletes who wish to rapidly increase muscle mass and performance. An increasing number of reports have indicated that nandrolone may affect and modulate the immune system. This study aimed to investigate the effects of nandrolone on septic shock-caused immune responses and the cellular mechanism of action using a sepsis murine model. Before septic shock induction, BALB/c mice were given a high dose of nandrolone or peanut oil only. After septic shock induction, mice were sacrificed at different time points. Their blood and tissue specimens were analyzed. It was found that the high-dose nandrolone group had significantly increased mortality compared with the control group (p < 0.001). The serum malondialdehyde level was significantly increased in the high-dose group compared with the control group. Animals administered a high dose of nandrolone had significantly increased hepatic tumor necrosis factor-a or splenic interferon-g at 0 and 6 hours. In lung tissue, insulin-like growth factor-1, insulin-like growth factor binding proteins (IGFBPs) and insulin-like growth factor-1 receptor, and IGFBP1 and IGFBP2 mRNA expression were increased in the high-dose nandrolone group at 6 hours. Nandrolone abuse may hasten the death of patients with septic shock and may aggravate septic shock in mice.

* Corresponding author. Comprehensive Breast Cancer Center, Changhua Christian Hospital, 135 Nanhsiao St, Changhua 500, Taiwan. E-mail address: [email protected] (D.-R. Chen). 1 Equal Contribution. 1607-551X/$36 Copyright ª 2011, Elsevier Taiwan LLC. All rights reserved. doi:10.1016/j.kjms.2010.12.015

Nandrolone aggravates septic shock

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摘要 諾龍（Nandrolone），一種人工合成的同化性類固醇，許多運動員都會用以快速增加肌肉 量及運動表現。越來越多報導指出諾龍可能會影響並調節免疫系統。本研究欲分析諾龍對敗血性 休克所引起的免疫反應之影響，並利用敗血性休克小鼠模式探討相關細胞作用機制。對BALB/c小 鼠誘發敗血性休克前，將實驗動物分成對照組及實驗組，每日分別給予高劑量花生油或諾龍。小 鼠誘發敗血性休克後，在不同時間點犧牲，採集血液及組織樣本進行分析。分析結果發現，實驗 組（高劑量諾龍）小鼠的死亡率明顯高於對照組（p＜0.001）；同樣地，實驗組血清malondialdehyde濃度也明顯較高。給予高劑量諾龍後0及6小時，實驗組動物的肝臟腫瘤壞死因子a或脾臟干 擾素g會明顯上升。實驗組接受高劑量諾龍後6小時，其肺臟組織的第一型類胰島素生長因子 （insulin-like growth factor-1）及其受體蛋白、類胰島素生長因子結合蛋白與類胰島素生長因子 結合蛋白1及2之mRNA表現都會增加。本研究結果證實諾龍會使小鼠的敗血性休克程度惡化，由 此推論諾龍的濫用會加速敗血性休克患者的死亡。 Copyright ª 2011, Elsevier Taiwan LLC. All rights reserved.

Introduction Nandrolone is an anabolic-androgenic steroid (AAS) derived from the male sex hormone testosterone. More than 40 derivatives can be taken orally or by injection. AAS is widely used by athletes to increase muscle mass. In the 1990s, many reports of AAS abuse among athletes and bodybuilders appeared [1]. They used a high dose of AAS to achieve a rapid increase in muscle mass and enhance athletic performance. However, anabolic steroid abuse is associated with many adverse effects with regard to the liver, cardiovascular function, endocrine and reproductive systems, and the musculoskeletal and central nervous systems [2e5]. Although AAS is often misused by people who want to increase muscle mass, it is also a useful therapeutic agent in treating a wide range of diseases. Elderly male patients who are on hemodialysis have improved nutrition after administration of nandrolone decanoate for more than 6 months. Nandrolone decanoate seems to be an acceptable treatment for anemia during dialysis [6]. Many reports on the effect of nandrolone therapy on HIV-infected patients have indicated a great increase in fat-free mass, and that nandrolone therapy may prove to be beneficial in reversing weight or lean tissue loss in HIV-infected patients and those with other chronic catabolic diseases [7e10]. However, there have been only a limited number of reports clarifying the effects of therapeutic levels of AAS on immune responses [11e14]. The effects of high-dose AAS on immunomodulation are unclear. Hughes et al. [2] used a murine model to mimic anabolic steroids abuse in humans and demonstrated that a high dose of anabolic steroids can affect immune responses. Nandrolone decanoate not only significantly affects cytokine production in human and murine cells but also inhibits the production of corticotropin in human peripheral blood lymphocytes. Some of those who abuse anabolic steroids suffer septic shock; however, whether nandrolone aggravates septic shock remains an open question. Despite many years of efforts in attempting to decrease the septic shock mortality rate, the results are generally disappointing [15e17]. Regarding to the cellular mechanism of nandroloneinduced muscle fiber hypertrophy and septic shock, Lewis et al. [18] demonstrated that nandrolone-induced diaphragm muscle fiber hypertrophy is mediated by the

influence of the insulin-like growth factor (IGF) family within the muscle, and coordinated changes in IGF binding proteins (IGFBPs) associated with an anabolic response [19e25]. In addition, Qiao et al. [19] reported that IGF-1 increases extracellular signal-regulated kinases 1/2 (ERK1/ 2) activation, a septic shock pathway. Liu et al. [20] pointed out that IGF-1 activates both phosphoinositide-3 kinase and ERKs, which contribute to the stimulation of nuclear factor kappa B (NF-kB)-dependent transcription, also a septic shock pathway. Furthermore, the NF-kB-responsive gene products are known to be free radicals, tumor necrosis factor-a (TNF-a), interferon-g (IFN-g), and IGFBPs. Lewis et al. [18] reported that the precise actions of IGFBPs expressed at the different tissue level are unknown and IGFBPs may exert effects independent of the IGF system. Nandrolone could have some effects on septic shock by activating these pathways in the liver, spleen, and lung through IGFBPs. Hughes et al. [2] also reported that nandrolone induced the production of TNF-a from human peripheral blood lymphocytes and inhibited IFN-g. They mentioned nandrolone abuser had deleterious effects of liver neoplasia, hepatic angiosarcoma, and hepatitis. It remains an open question whether IGF-1 and IGFBPs play a role on the septic shock pathways, TNF-a, and IFN-g. In this study, we used an animal model to mimic nandrolone abuse and to determinate the effects on immune responses after septic shock induction. We hypothesized that high-dose nandrolone increases IGF-Type 1 receptor (IGF-1R) expression, subsequently modulating innate immunity NF-kB-responsive gene transcription and increasing free radical, TNF-a, IFN-g, and IGFBPs production.

Material and methods Animals Male BALB/c inbred mice were purchased from the National Laboratory Animal Breeding and Research Center (Taipei, Taiwan) and housed individually in the Laboratory Animal Center, Changhua Christian Hospital (Changhua, Taiwan) with food and water available ad libitum. They were kept on a 12:12 hour light/dark cycle. All studies were initiated with male BALB/c mice that were approximately 6 weeks of age. All surgical procedures and care administered to the

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animals in this study were approved by the Laboratory Animal Care and Use Committee of Changhua Christian Hospital, Changhua, Taiwan.

Induction of septic shock Septic shock is defined as a serious medical condition caused by decreased tissue perfusion and oxygen delivery as a result of infection and sepsis; the immune systems of septic shock patients cannot deal with infection as effectively as those of healthy adults [17]. For this study, Escherichia coli ATCC25922 (ATCC, Rockville, MD, USA) was cultured in Luria-Bertani broth (LB, Difco, Detroit, MI, USA) at 37
C and harvested at the mid-log phase for inoculation. The concentration of bacteria was determined with a spectrophotometer with an optical density (600 nm) of 1 equal to 108 colony forming unit (CFU)/mL. Mice were inoculated intraperitoneally with 7  108 CFU of E coli in sterile saline to induce septic shock. The inoculums were plated on nutrient agar plates and incubated at 37
C for 24 hours to determined viable counts.

Nandrolone administration Nandrolone-phenylpropionate (nandrolone; China Chemical & Pharmaceutical Co. Ltd., Taipei, Taiwan) was dissolved in peanut oil. In a survival assay, 24 BALB/c mice were separated into three groups. Group one (n Z 8) received lowdose nandrolone (0.025 mg/mouse), group two (n Z 8) received high-dose nandrolone (0.25 mg/mouse), and group three (n Z 8) received peanut oil only (5 mL/kg) by intraperitoneal injection, respectively, once a day for 14 consecutive days before infection with bacterial inoculation. The doses of nandrolone were estimated according to a previous study [18], and the high-dose group was defined as the nandrolone abuse group in this study. For the assay of the effect of nandrolone abuse on pathophysiological features, 24 BALB/c mice were randomly separated into two groups. One group (n Z 12) was injected intraperitoneally with high-dose nandrolone (0.25 mg/ mouse/d), and the other group (n Z 12) received isovolemic

Table 1

peanut oil by intraperitoneal injection for 14 days. On the 14th day, 1 hour after the completion of treatment, all mice were inoculated with E coli (7  108 CFU/mouse) by intraperitoneal injection and were sacrificed at 0, 3, or 6 hours after E coli challenge.

Determination of numbers of viable bacteria Mice were sacrificed by inhalation of ether; and the abdominal cavity, blood, liver, spleen, and lung were obtained aseptically. The organs were homogenized with a cell strainer (BD Falcon, Bedford, MA, US) in sterile saline. The abdominal cavities, portions of blood, and organ homogenates were placed on nutrient agar plates and cultured at 37
C for 24 hours to determine the E coli burden. The remaining blood was collected in heparinized tubes, centrifuged at 1,500 revolutions per minute (rpm) for 10 minutes at 4
C to obtain the plasma, and then stored at 80
C until use.

Determination of cytokines in organ homogenates The remaining organ homogenates were centrifuged at 3,000 rpm for 15 minutes at 4
C, and the supernatants were collected and stored at 80
C until assay. The TNF-a level in supernatants of liver homogenate and the IFN-g level in supernatants of spleen homogenate were measured with a commercial enzyme-linked immunosorbent assay kit (Biosource, Camarillo, CA, USA).

Determination of the malonaldehyde concentration in serum Lipid peroxidation parameter malonaldehyde (MDA) is a well-known index of free radical activity. For the measurement of MDA, we used thiobarbituric acid, which measures MDA-reactive products. Whole blood samples were collected in serum separator tubes, allowed to clot for 30 minutes, centrifuged at 2,000 rpm for 15 minutes at room temperature, and stored at 80
C until assayed. Then, 0.5 mL serum and 2.5 mL trichloroacetic acid were

Primers used to detect real-time polymerase chain reaction

Genes mouse b-actin mouse IGF-I mouse IGF-1R mouse IGFBP1 mouse IGFBP2 mouse IGFBP3

Primer sequences 0

Amplicon size (bp) 0

5 -TTGTGATGGACTCCGGAGAC-3 50 -TCATCACTATTGGCAACGAGC-30 50 -GGTTTATGAATGGTTCCCTTATCT-30 50 -GCCACTTGACTACTTATTTGCTAT-30 50 -CAAGCT GTGTGTCTCCGAAA-30 50 -TGATTCGGTTCTTCCAGGTC-30 50 -GCCGACCTCAAGAAATGGAA-30 50 -GTAGACACACCAGCAGAGT-30 50 -CCCGAGTGCCATCTCTTCTA-30 50 -GCTCAGTGTTGGTCTCTTTC-30 50 -GACACCCAGAACTTCTCCTC-30 50 -CATACTTGTCCACACACCAGC-30

bp Z base pairs; IGF-1R Z insulin-like growth factor type 1 receptor; IGFBP Z insulin-like growth factor binding proteins.

309 167 161 198 200 219

Nandrolone aggravates septic shock

Real-time polymerase chain reaction Reverse transcription was performed using a kit for cDNA synthesis (Takara, Kyoto, Japan). Reactions were run on a LightCycler 3.0 (Roche, Penzberg, Germany). Thermal cycling conditions consisted of initial steps of 15 seconds at 95
C, followed by 72
C for 30 seconds, for a total of 40 cycles. All data were normalized to b-actin mRNA levels to account for any variation in RNA concentrations between samples. The expressions of IGF-1R and IGFBPs in the lungs were quantitatively examined by real-time polymerase chain reaction (PCR) with the TaqMan-probed real-time PCR assay (Roche Diagnostic, Germany). The mouse b-actin was used as an endogenous control gene. The primer pairs used for IGF-1R and IGFBPs gene transcripts are shown in Table 1. The amplification of the target genes was as follows: 95
C for 10 minutes, followed by 50 cycles of 95
C for 15 seconds, and 60
C for 1 minute. Each sample was calculated in three different experiments.

Statistical analysis The effects of intervention were first tested by directly comparing the nandrolone and control groups. Data in the tables, figures, and text were expressed as mean  standard deviation. The difference between groups was determined using a two-tailed unpaired Student t test (for means of continuous variables), as indicated. A p value of less than 0.05 was considered statistically significant, based on a 0.9 power. Statistical analyses were performed using SPSS software (SPSS version 10.0; SPSS Inc., Chicago, IL, USA).

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mixed and centrifuged at 3,300 rpm for 10 minutes. The pellet was collected, rinsed with 2.5 mL 10% acetic acid, and resuspended in 2.5 mL 10% acetic acid. After adding 0.2% thiobarbituric acid (in 2 mol/L Na2SO4), the mixture was mixed, heated at 100
C for 30 minutes, and transferred onto ice immediately. MDA was then extracted with 2 mL nButanol. The upper layer was collected and the absorption was measured at 531 and 560 nm. A Model UV-240 (Shimazu, Kyoto, Japan) double-beam UV visible recording spectrophotometer, equipped with the Model OP 1-2 option program, was used for zero-order and second-derivative measurements. Scans were obtained with a 2-nm bandwidth, a scan speed of 180 nm/min, and an absorbance scale of 0.02e0.2 nm (full scale). The second-derivative spectrum of MDA was measured from 500 to 600 nm in a 1-cm (1 mL) quartz cuvette against a freshly prepared reagent blank. Each sample was measured four times to ensure the precision of measurement. The peak amplitude at 531 and 560 nm with respect to the adjacent satellite peak (at a longer wavelength) was measured. A standard curve was prepared by treating standards in the same manner, and sample concentrations were calculated according to this curve. The difference in absorbance between 531 and 560 nm was measured; the MDA concentration was calculated from a calibration curve of serial standards and corrected by multiplying the dilution factor (the final volume of the aqueous layer).

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Time after septic shock induction (hour) Figure 1. Survival curve, the effect of nandrolone on subsequent septic shock. Mice were divided into three groups: the control group was given peanut oil only; the low-dose and high-dose nandrolone groups were given 0.025 mg/mouse and 0.25 mg/mouse, respectively for 14 days. A significant difference (p Z 0.001) in survival was observed in the high-dose nandrolone-treated mice (0.25 mg/mouse, intraperitoneally [i.p.], n Z 8) compared with the control mice (peanut oil, 0.1 mL/mouse, i.p., n Z 8). No significant decrease (p > 0.05) in survival was observed in the low-dose nandrolone-treated mice (0.025 mg/mouse, i.p., n Z 8) compared with the control mice.

Results To explain whether nandrolone abuse is associated with mortality in the user who suffers septic shock, we defined the high-dose nandrolone group as the nandrolone abuse group. Animals administered high-dose nandrolone had significantly increased mortality compared with the control group (Fig. 1, p < 0.001), but there was no effect on the survival rate of the low-dose nandrolone group and the control group. These data indicate the dose of nandrolone was associated with mortality, especially during septic shock. Use of high-dose nandrolone may hasten mortality because of septic shock. The bacterial burden may be linked to increased mortality in high-dose nandrolone-treated mice during septic shock. We examined the bacterial load in the abdominal cavity fluid, whole blood, liver, and spleen and found no significant difference between the high-dose mice and the control mice (Fig. 2). However, many pathophysiological changes were observed during severe sepsis and septic shock, and one of the most striking was metabolic derangement. Hyperglycemia was the most important of the metabolic changes [26,27]. We measured the glucose level in the blood at 0, 3, and 6 hours after E coli inoculation. The blood glucose in the high-dose nandrolone group showed no significant difference compared with the control group at 0 and 6 hours (Fig. 3A). These data indicate

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Figure 2. Effect of high-dose nandrolone on the bacterial burden. The effect of high-dose nandrolone (0.25 mg/mouse/d, intraperitoneally [i.p.], for 14 days) on the bacterial burden of the (A) abdominal cavity; (B) whole blood; (C) liver; and (D) spleen of mice inoculated with Escherichia coli ATCC 25922. The bacterial burden count in the abdominal cavity, whole blood, liver, and spleen is shown as CFU/mL, mean  standard deviation. No significant increase (p > 0.05) in the bacterial burden was observed in the abdominal cavity fluid, and in the whole blood, liver, and spleen of high-dose nandrolone-treated mice compared with the control mice. CFU Z colony forming units.

that the increased mortality with use of high-dose nandrolone during septic shock was not significantly related to the bacterial load or metabolic change. The effect of nandrolone on mortality may manifest through other pathways. MDA is an end product produced from the peroxidation of cell membrane lipid, and lipid peroxidation is triggered by a variety of proinflammatory mediators [28,29]. Serum MDA levels were assessed as markers of lipid peroxidation and hence oxidative stress. We measured the serum MDA levels at 0, 3, and 6 hours after septic shock induction. Mice receiving high-dose nandrolone had significantly increased MDA levels in serum compared with the control group, which was given peanut oil only (Fig. 3B). In addition, we determined the proinflammatory cytokine production; hepatic TNF-a and splenic IFN-g were significantly increased at 0 and 6 hours (Fig. 3C and D). These data indicated use of high-dose nandrolone influences immunomodulation during septic shock by increasing proinflammatory cytokine production and enhancing peroxidant production by lipid peroxidation.

To determine whether IGF-1R is the mediator involved in the modulation of high-dose nandrolone in sepsis, we measured the expression of lung IGF-1R and IGFBPs mRNAs using real-time reverse transcription-PCR. Compared with 0 hour, we demonstrated that high-dose nandrolone increased lung IGF-1R, as well as IGFBPs mRNA expression, at 6 hours (Fig. 4). Neither increase of IGFBP2 expression nor decrease of IGFBP3 expression were found (p > 0.05). These data indicated that high-dose nandrolone influences IGF-1R and IGFBPs in septic shock.

Discussion To our knowledge, this is the first report on high-dose nandrolone, an AAS that aggravates septic shock in a murine model. This study shows that high-dose nandrolone increases serum-free radical parameter MDA production, hepatic TNF-a, splenic IFN-g, and lung IGF-1R, as well as IGFBPs mRNA expression, which may modulate the pathogenesis of septic shock.

Nandrolone aggravates septic shock

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Figure 3. Effect of high-dose nandrolone on blood, liver, and spleen. The effect of high-dose nandrolone (0.25 mg/mouse/d, intraperitoneally [i.p.], for 14 days) on (A) glucose in the blood; (B) MDA in the blood; (C) TNF-a of the liver; and (D) IFN-g of the spleen in mice inoculated with Escherichia coli ATCC 25922. The data are shown as mean  standard deviation. No significant decrease (p > 0.05) of glucose was observed at 0 and 6 hours in the blood of the high-dose nandrolone-treated mice compared with the control mice. High-dose nandrolone increased serum MDA levels at 0, 3, and 6 hours. High-dose nandrolone significantly increased liver TNF-a levels at 0 and 6 hours (C) and spleen IFN-g levels at 0 and 6 hours (D). IFN-g Z interferon-g; MDA Z malonaldehyde; TNF-a Z tumor necrosis factor-a.

Our data demonstrated that many of the variable baselines differed in the high-dose nandrolone-treated and control mice at 0 hour. This implies that high-dose nandrolone, and not septic shock, is related to the baselines. In addition, our results are consistent with those of a previous study [18] that reported that there was a significant increase in metabolic imbalance after nandrolone therapy. Nandrolone abuse results in a hormonal and metabolic imbalance that impacts surrogate variables. That is also why we used male mice in this study to reduce the menstrual impact on hormonal and metabolic imbalance. However, we neither have precise statistics on how often nandrolone abusers die from septic shock nor how long the effects of AAS persist following cessation. In this study, there was no cessation for more than 24 hours. Our data showed that free radical parameter serum MDA is enhanced by high-dose nandrolone. This is in accordance

with the study by Green et al. [30] that reported that nandrolone induces changes in the behavior of the nitric oxide dilator system in the blood flow responses of the human brachial artery. On the other hand, Ferrer et al. [31] found that nandrolone inhibited nitric oxide production in rabbit aorta endothelium. We confirmed that high-dose nandrolone increases hepatic TNF-a at 0 and 6 hours. This finding is in accordance with that of a previous study. Hughes et al. [2] found that nandrolone directly induces the production of the inflammatory cytokines TNF-a and interleukin-1 b (IL-1 b) from human peripheral blood lymphocytes, but has no effect on IL-2 or IL-10 production. Modulating peripheral blood lymphocytes, then hepatic TNF-a, sequentially, might be the pathway through which high-dose nandrolone modulates the liver response in septic shock. Contrary to our supposition, our data demonstrated that high-dose

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Figure 4. Effect of high-dose nandrolone on lung IGF-1R and IGFBPs. The folds were defined as the ratios of the 6-hour realtime PCR value to 0 hour. High-dose nandrolone induction of IGF-1R and IGFBP1 mRNA expression in lung tissue in vivo. BALB/c mice were injected with a vehicle or high-dose nandrolone (0.25 mg/mouse/d for 14 days) intraperitoneally. Mice were sacrificed before (0 hour) and 6 hours after Escherichia coli ATCC25922 administration. The data are shown as mean  standard deviation. *Folds of high-dose nandrolone were significantly different from control (p < 0.05). IGF-1R Z insulin-like growth factor type 1 receptor; IGFBP Z insulin-like growth factor binding proteins; PCR Z polymerase chain reaction.

nandrolone increases splenic IFN-g levels at 0 and 6 hours. Thus, IFN-g may be another pathway that high-dose nandrolone uses to modulate the pathogenesis of septic shock. In contrast, Hughes et al. [2] reported that nandrolone inhibited IFN production in human WISH and murine L-929 cells. Meanwhile, in this study the blood glucose in the high-dose nandrolone group showed no significant difference compared with the control group at 0 and 6 hours (Fig. 3A). The interaction of TNF-a, IFN-g, and blood glucose remained unclear in this study. Nandrolone may cause TNF-a and IFN-g upregulations, which did not mean this could be the side effect. This phenomenon may reflect the protection mechanism in the immune system. On the other hand, immune cells (such as macrophage, dendritic cell, and lymphocytes) and endothelial cells are known to play the key roles in the regulation of the pathophysiological process of sepsis through the proinflammatory mediators (e.g. IL-1, nitric oxide) and anti-inflammatory mediators (e.g. IL-10) [32]. Further study of the mediators might be necessary to clarify the cellular mechanism in vivo and in vitro. High-dose nandrolone increased lung IGFBP1 and IGFBP2 in this study, but decreased lung IGFBP3; this result contrasts with that of a previous study, in which nandrolone was shown to increase IGFBP3 in the diaphragm (Lewis et al. [18]). However, there are potential limitations to this study. First, whether or not IGF-1R was induced by highdose nandrolone is still questionable. The study also does not clearly address the tissue-specific differences. The liver is supposed to be the main source of IGF-1. Further study is necessary to clarify the problem. Lung ERKs and NF-kB might provide us with some information regarding

modulation in the lung. Second, this study does not take into account the fact that the ERKs pathway modulates the cell cycle. The IGF-1R-ERKs-AP-1-cdk/cyclin-cell proliferation pathway and other ERKs pathways might contribute to septic shock pathogenesis [33]. Whether high-dose nandrolone influences the cell cycle is unclear. Most of the IGF-1 in the blood is associated with a 150-kD complex consisting of IGF-I itself (g subunit), glycosylated IGF binding protein-3 (IGFBP3, b subunit), and a glycosylated acid-labile protein (a subunit). Reed et al. [34] reported that a decrease in IGF-1 has been seen in patients with breast cancer treated with tamoxifen. The mechanisms by which circulating IGF-1 levels are decreased reduce hepatic IGF-I synthesis and attenuate growth hormone action by increasing growth hormone-binding protein production. However, our results substantiated that high-dose nandrolone increases lung IGFBP1 as well as IGFBP2 (Fig. 4) and decreases lung IGFBP3. On the other hand, all forms of IGFBPs have a high affinity for IGF-1. Each binding protein has unique features, including posttranslational modifications, tissue distribution, and the ability to enhance or attenuate IGF-1 actions. Pannier et al. [35] reported that IGFBP3 mRNA is weakly expressed in fetal kidney. Although the steady-state level of IGFBP3 mRNA is low, its rate of translation may be high, resulting in relatively high levels of its IGFBP3 protein that are eligible for posttranslation modulation. Modulation could occur at the posttranslational stage for IGFBP3 in the lung. Nandrolone has been reported to decrease intramuscular branched-chain amino acids in trauma patients [36]. Furthermore, enteral hyperalimentation with an elemental diet (e.g. Vivonex) was reported by Moss et al. [37] to elevate serum branched-chain amino acid (leucin, isoleucine, and valine) levels. In this study, we also observed those mice receiving high-dose nandrolone demonstrated muscle hypertrophy that might point out the abuse effects. However, we did not have precise statistics on how strong high-dose nandrolone induced muscle hypertrophy. It is one of potential limitations of this study. In conclusion, it is suggested that use of high-dose nandrolone may aggravate septic shock and increases the serum MDA level, hepatic TNF-a, splenic IFN-g, and lung IGF-1R, as well as lung IGFBPs mRNA expression in mice. Nandrolone may have the serious potential side effect of septic shock for its abuser.

Acknowledgments This work was supported by grants NSC97-2314-B371-006 from the National Science Council (Taipei, Taiwan) and 97-CCH-IPI-44 from Changhua Christian Hospital (Changhua, Taiwan).

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