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Glucocorticoids Suppress the Inflammation-Mediated Tolerance to Acute Toxicity of Cadmium in Mice
Toxicology and Applied Pharmacology 178, 1–7 (2002) doi:10.1006/taap.2001.9323, available online at http://www.idealibrary.com on
Glucocorticoids Suppress the Inflammation-Mediated Tolerance to Acute Toxicity of Cadmium in Mice Kyong-Son Min, 1 Heisuk Kim, Miho Fujii, Noriko Tetsuchikawahara, and Satomi Onosaka Department of Nutrition, Kobe Gakuin University, Ikawadani-cho, Nishi-ku, Kobe, 651-2180, Japan Received April 13, 2001; accepted October 15, 2001
parenterally at high doses, Cd 2⫹ can induce lethal hepatocellular necrosis as well as extensive testicular lesions (Sauer et al., 1997a; Rikans and Yamano, 2000). It has been demonstrated that Cd 2⫹ induces multiple cytotoxic and/or metabolic effects, such as interference with the action of essential metals (Chmielnicka and Sowa, 1996) and alteration of the activity of various enzymes (Kinne-Saffran et al., 1993; Casalino et al., 1997; Patra et al., 1999). Hepatic injury due to Cd exposure can involve both direct and indirect damage to the hepatocyte, as seen with some other xenobiotics. Increased expression of several cytotoxic mediators, including tumor necrosis factor (TNF)-␣, has been demonstrated in rodent liver or hepatocytes following acute Cd 2⫹ exposure (Kayama et al., 1995a,b; Dong et al., 1998). It has been demonstrated that Kupffer cell inhibitors or enhancers modulate the levels of hepatotoxicity induced by certain xenobiotics (Edwards et al., 1993; El Sisi et al., 1993; Blazka et al., 1995; Michael et al., 1999). However, either its inhibitors or enhancers resulted in the attenuation of Cd-induced hepatotoxicity (Sauer et al., 1997a,b). The mechanism of the tolerance to Cd is thought to be mediated by the induction of hepatic metallothionein (MT). These data suggest that cytokines might demonstrate a preference for the protective effect by hepatic MT induction over the cytotoxic effects on progression of Cd-induced liver injury. Therefore, sequestration of Cd by MT in the early stages, post-Cd dosing, is the most effective mechanism for decreasing Cd toxicity. Several compounds, including metals, steroid hormones, calmodulin inhibitors, antioxidants, and inflammatory agents have been shown to promote tolerance to Cd-induced injury by inducing synthesis of MT, either directly or indirectly (Goering and Klaassen, 1984a,b; Shiraishi et al., 1993, 1994; Sauer et al., 1997b). The proposed mechanism of this MT-mediated Cd-tolerance involves sequestering Cd 2⫹ from molecular targets by high-affinity binding of MT to Cd 2⫹. MT is a small, cysteine-rich, metal-binding protein that plays a role in zinc homeostasis, heavy metal detoxification, and the protection of cells against reactive oxygen intermediates and electrophilic anticancer drugs. The rapid induction of MT gene transcription by heavy metals is mediated through metal response elements, which are present in multiple copies in the proximal promoters
Glucocorticoids Suppress the Inflammation-Mediated Tolerance to Acute Toxicity of Cadmium in Mice. Min, K.-S., Kim, H., Fujii, M., Tetsuchikawahara, N., and Onosaka, S. (2002). Toxicol. Appl. Pharmacol. 178, 1–7. Several compounds have been shown to cause acute toxicity to cadmium (Cd). The mechanism of tolerance to Cd toxicity induced by glucocorticoids or by inflammation involves induction of metallothionein (MT) synthesis via glucocorticoid response elements or by inflammatory cytokines. We have demonstrated previously that the synthetic glucocorticoid dexamethasone suppresses inflammation-mediated induction of hepatic MT synthesis. Here we investigated the effect of glucocorticoid on tolerance to Cd induced by inflammation in mice. The LD50 of Cd for mice with induced inflammation by injection with turpentine oil (Turmice) was higher than the LD50 in control mice. Pretreatment of Tur-mice with dexamethasone to the Tur-mice (DexⴙTur-mice) resulted in a decrease in LD50 after Cd treatment. A significant increase in plasma alanine aminotransferase and aspartate aminotransferase levels in the DexⴙTur-mice was observed at lower doses of Cd than in the Tur-mice and at higher doses of Cd than in control mice. Dexamethasone did not suppress tolerance to cadmium toxicity in the testes of the Tur-mice. Pretreatment of Tur-mice with dexamethasone resulted in suppression of both plasma interleukin (IL)-6 elevation and in suppression of hepatic MT levels when induced by inflammation but not when induced by Cd. These data suggest that suppression of tolerance to Cd toxicity induced by glucocorticoid may involve hepatic MT synthesis mediated by inflammatory cytokines, such as IL-6. We suggest that the inflammatory response can modulate Cd toxicity by induction of MT by inflammatory cytokines. © 2002 Elsevier Science Key Words: cadmium; inflammation; glucocorticoid; inflammatory cytokine; tolerance.
Cadmium (Cd) is a widespread environmental pollutant that poses a significant health risk to humans and animals. It has been found that Cd 2⫹ can produce both acute and chronic tissue injury and can damage various organs including the lung, liver, kidney, bone, testis, and placenta, depending on the dose, route, and duration of exposure (for reviews, see Goering et al., 1995; Jarup et al., 1998). For example, when administered 1
To whom correspondence should be addressed. 1
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of the MT genes. MT-I and MT-II are acute phase proteins induced in response to a variety of physiological and pharmacological agents such as glucocorticoids, inflammatory compounds, and interleukins. Glucocorticoid directly induces MT synthesis via the glucocorticoid response elements (GRE). Induction of MT synthesis by inflammation following injection of turpentine oil, n-hexane, or lipopolysaccharide (LPS) has been shown to be mediated through inflammatory cytokines (De et al., 1990; Min et al., 1991; Itoh et al., 1994, 1996). We have shown previously that glucocorticoid directly increases hepatic MT synthesis in mice and can also suppress inflammation-induced increases in MT (Min et al., 1992). In contrast, in both animal and cell culture studies, dexamethasone induced MT synthesis synergistically with increases in IL-6 (Sato et al., 1996; Kasutani et al., 1998). We suggest that glucocorticoid may both inhibit the production of cytokines and may stimulate cytokine-mediated induction of MT, resulting in glucocorticoid having two opposing functions in the induction of hepatic MT. Moreover, inflammatory cytokines are involved in Cd hepatotoxicity. Here we have investigated the effects of glucocorticoids on inflammation-induced Cd toxicity in mice.
FIG. 1. Effect of pretreatment with Tur and/or Dex on acute lethal toxicity of Cd at various doses. CdCl 2 (0.2–3.0 mg Cd/kg) was injected to pretreated mice (0 – 0.6 mg Cd/kg; 12 mice/each dose, 0.8 –3.0 mg Cd/kg; 16 mice/each dose) with saline, turpentine oil (Tur, 10 ml/kg sc), and/or dexamethasone (Dex, 25 mg/kg sc). Survived number was counted after 48 h. Control, nonpretreated mice; Tur, Tur-pretreated mice; Dex⫹Tur, pretreatment with Tur to Dex-pretreated mice.
MATERIALS AND METHODS Animals. Male mice (ddY strain) 6 weeks old and weighing approximately 30 g were purchased from Nihon SLC (Shizuoka, Japan). Mice were maintained on a 12-h light– dark cycle and given food and water ad libitum (Type MF, Oriental Yeast, Osaka, Japan). All animal experiments were carried out under the control of the Animal Research Committee, in accordance with the Guidelines on Animal Experiments in Kobe Gakuin University and Japanese Government Animal Protection and Management Law (No. 105). Chemicals. All chemicals were purchased from Nakarai Tesque, Inc. (Kyoto, Japan). Pretreatment of mice with turpentine oil and/or dexamethasone. Two hundred milligrams of dexamethasone (Dex), a synthetic glucocorticoid, was suspended in 10 ml of 5% acacia (Arabic gum) solution. Typically, 24 h following pretreatment with Dex (25 mg/kg) or its vehicle, the mice were treated subcutaneously with 10 ml/kg turpentine oil (Tur) or saline. After an additional 24 h, the animals received a single dose of CdCl 2 (0.2–3.0 mg Cd/kg iv) in 10 ml/kg saline via the femoral vein and either were observed or were euthanized by overanesthesia at the indicated time. Heparinized blood was collected from the posterior vena cava 16 h after injection of Cd. Sections of liver and kidney were homogenized with 0.25 M sucrose solution and then were analyzed for both Cd and MT concentrations. Testes were taken 48 h post-Cd injection and were homogenized with Tris buffer (10 mM, pH 7.6) for analysis of testicular hemoglobin concentrations. Plasma activities of hepatic enzymes. Plasma activities of both alanine aminotransferase (ALT) and aspartate aminotransferase (AST) were determined spectrophotometrically using Transaminase CII Wako kits (Wako Pure Chemical Industries, Ltd., Osaka, Japan). Absorbance of hemoglobin. To quantify the extent of cadmium-induced testicular hemorrhage, hemoglobin levels in the 18,000g supernatant fraction from testicular homogenates were determined by measuring absorbance at 414 nm by the method of Niewenhuis and Prozialeck (1987). Analysis of Cd concentration. Following overnight digestion of the tissue homogenates with nitric acid, the solutions were brought to a final volume of 4 ml with water and were filtered using a 0.45-m Dismic-13CP syringe tip
filter. Samples were analyzed for Cd 2⫹ concentration using a flame atomic absorption spectrophotometer (Z-5000 Polarized Zeeman Atomic Absorption Spectrophotometer, Hitachi, Tokyo, Japan). Determination of MT. Hepatic MT concentrations were determined by the Cd– heme method as described previously (Onosaka et al., 1978). Measurement of plasma IL-6 and TNF-␣. The concentrations of interleukin-6 (IL-6) and tumor necrosis factor-␣ (TNF-␣) in plasma were measured by ELISA. Purified rat anti-mouse IL-6 and the TNF-␣ monoclonal antibodies were obtained from Fujisawa Pharmaceutical Co. Ltd. (Pharmigen, San Diego, CA). Recombinant mouse IL-6 and TNF-␣ obtained from Fujisawa Pharmaceutical Co. Ltd. (Pharmigen) were used as standards. Statistics. Results are expressed as means ⫾ SD. Data were analyzed for significance by Student’s t test and by a two-tailed ANOVA. Differences were considered significant at p ⬍ 0.05.
RESULTS
Effect of Pretreatment with Tur and/or Dex on the Lethal Toxicity of Cd The Tur and/or Dex-pretreated mice were observed 24 h following administration of increasing doses of Cd (Fig. 1) and survival rates (%) were calculated. The LD50 of Cd was 1.25 mg Cd/kg for control mice. When mice were given an injection of turpentine oil to initiate inflammation (Turmice), the LD50 for Cd increased to 2.5 mg Cd/kg. However, in mice pretreated with Dex 24 h prior to injection with Tur (Dex⫹Tur-mice), the LD50 for Cd was 2 mg Cd/kg, lower than in the Tur-treated mice but higher than in control mice. These data suggest that initiation of inflammation
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FIG. 2. Effect of pretreatment with Tur and/or Dex on Cd hepatotoxicity. Activities of aminotransferases (ALT and AST) were determined at 16 h after an injection of Cd. Data points represent means ⫾ SD (n ⫽ 4–8). *Significantly different from no Cd-injected mice (0 mg Cd/kg), p ⬍ 0.05.
resulted in a tolerance to Cd toxicity and that glucocorticoid suppresses this tolerance. Effect of Pretreatment with Tur and/or Dex on Cd-Induced Hepatotoxicity and Testicular Toxicity Mice administered CdCl 2 demonstrated clear signs of liver injury, as measured by plasma levels of ALT and AST 16 h postdosing (Fig. 2). Pretreatment of the mice with Tur (Turmice) and/or Dex (Dex⫹Tur-mice) significantly attenuated the observed hepatotoxicity at a Cd dose of 1 mg/kg. Significant increases in plasma transaminase activities in the Dex⫹Turmice were observed at higher doses of Cd than in control mice and at lower doses of Cd than in Tur-treated mice. After a Cd injection at a dose of 1.5 mg/kg, no overt morphological alterations were associated with Cd in Tur-mice, but the hepatocellular necrosis was observed in Dex⫹Tur-mice (data not shown). Cd injection resulted in an increase in testicular hemoglobin after 48 h (Fig. 3), an indication of Cd-induced testicular hemorrhage. A significant increase in testicular hemoglobin concentration occurred at 1 mg Cd/kg in control mice. Testicular hemoglobin concentrations were significantly elevated in the Tur-mice and Dex⫹Tur-mice at Cd doses greater than 1.5 mg Cd/kg. Therefore, turpentine-induced inflammation seems to reduce the sensitivity of mice to acute Cd effects, such as hepatotoxicity and testicular toxicity, and glucocorticoid can decrease the inflammation-mediated tolerance to hepatotoxicity but does not affect testicular toxicity.
FIG. 3. Effect of pretreatment with Tur and/or Dex on testicular Cd toxicity. Testicular hemoglobin concentrations were measured at 48 h after an injection of CdCl 2 . Data points present means ⫾ SD of five mice. *Significantly different from no Cd-injected mice (0 mg Cd/kg), p ⬍ 0.05.
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FIG. 4. Effect of pretreatment with Tur and/or Dex on hepatic and renal Cd concentration. The concentrations of Cd were determined after 16 h. Data points represent means ⫾ SD (n ⫽ 4–8). a Significantly different from no Cd-injected mice, p ⬍ 0.05. b Significantly different from Tur-injected mice, p ⬍ 0.05.
Effect of Pretreatment with Tur and/or Dex on Hepatic and Renal Cd Retention Cd concentrations were measured in liver and kidney, tissues that typically retain Cd, 16 h following an injection of Cd (Fig. 4). Hepatic Cd accumulation following the dose of 1 mg Cd/kg in both Tur-mice and Dex⫹Tur-mice was higher than in the nonpretreated control mice. No significant difference in hepatic Cd accumulation between the Tur-mice and the Dex⫹Tur-mice was observed except at the 1–1.25 mg Cd/kg doses. In contrast, renal accumulation of Cd was significantly reduced in the Tur-mice compared to control mice at 1 mg Cd/kg. In addition, renal Cd accumulation was higher in Dex⫹Tur-mice than in the Tur-mice. The contrast in hepatic and renal distributions indicates that a reduction of Cd accumulation by extrahepatic tissues, such as in the kidneys, is offset by increased Cd accumulation by the liver. Effect of Pretreatment with Tur and/or Dex on Hepatic MT Concentration before or after Cd Injection In the liver samples used for analysis of Cd levels, hepatic MT concentrations 16 h following Cd injection were determined and are shown in Fig. 5. In agreement with a previous report from our laboratory (Min et al., 1992), treatment with Tur caused a significant increase in hepatic MT over control and this effect was suppressed by pretreatment of the mice with Dex before an injection of Cd. Hepatic MT concentrations in the Tur-treated mice significantly increased at all doses of Cd examined. Pretreatment of the Tur-mice with Dex resulted in a
FIG. 5. Effect of pretreatment with Tur and/or Dex on hepatic MT concentration after an injection of Cd. Hepatic MT concentrations were determined at 16 h after an injection of Cd. a Significantly different from no Cd-injected mice, p ⬍ 0.05. b Significantly different from Tur-injected mice, p ⬍ 0.05.
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FIG. 6. Effect of preinduced MT by Dex at various doses on hepatotoxicity of Cd. (A) Effect of preinduced MT by Dex (0 –100 mg/kg sc) without Tur on hepatotoxicity of Cd (1 mg Cd/kg sc). (B) Effect of preinduced MT by Dex (0 –100 mg/kg sc) with Tur on hepatotoxicity of Cd (1.5 mg Cd/kg sc). Hepatic MT concentrations were determined before an injection of Cd. Plasma ALT activities and testicular hemoglobin concentrations were determined at 16 and 48 h after a subsequent Cd injection, respectively. Data points represent means ⫾ SD (n ⫽ 4). *Significantly different from Dex (0 mg/kg) mice, p ⬍ 0.05.
slight suppression of these increases in hepatic MT. Combined, these results indicate that glucocorticoid can suppress hepatic induction of MT that results from Tur-initiated inflammation, but glucocorticoid does not alter subsequent Cd-induced changes in MT levels. Effect of MT Preinduction by Dex with or without Tur on Cd Toxicity Treatment with Dex alone resulted in a significant increase of hepatic MT concentrations independent of dose and caused dimunition of plasma ALT elevation following injection of Cd at a dose of 1 mg/kg (Fig. 6A). An injection of Dex alone did not affect plasma ALT activity before Cd injection (data not shown). The effects of the pretreated dose of Dex in Tur-mice on hepatic MT concentration before an injection of Cd, plasma ALT activity, and testicular hemoglobin concentrations are shown in Fig. 6B. Treatment with Tur alone (Dex 0 mg/kg) resulted in a remarkable increase of hepatic MT concentrations and diminished plasma ALT elevation following injection of Cd at a dose of 1.5 mg/kg (Fig. 6B). In contrast with pretreat-
ment of control mice with Dex (Fig. 6A), pretreatment of Tur-mice with Dex resulted in significant decreases of hepatic MT concentrations at a dose of 25–100 mg/kg. According to the significant decrease of hepatic MT concentration, pretreatment of the Tur-mice with Dex did not diminish Cd-induced ALT elevation. Finally, Dex pretreatment did not relapse Cdinduced testicular toxicity in these mice, as seen by the lack of changes in testicular Hb levels. Effect of Pretreatment with Dex on Plasma Concentration of IL-6 and TNF-␣ after an Injection of Tur IL-6 and TNF-␣ levels were determined following injection of mice with Tur, in the absence or presence of Dex pretreatment (Fig. 7). There was a marked elevation in plasma IL-6 level 16 h following injection with Tur. Although both Dex and Tur treatments alone resulted in significant increases in plasma IL-6 concentrations, pretreatment with Dex to Tur-mice resulted in IL-6 levels that were similar to those seen with Dex only. Tur treatment did not result in elevated plasma TNF-␣. These results indicate that the Tur-initiated inflammation
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FIG. 7. Effect of Dex on plasma concentration of cytokines following inflammation initiated by Tur. (A) Plasma concentration of IL-6 and TNF following inflammation initiated by Tur (10 ml/kg sc). (B) Effect of pretreatment with Dex (25 mg/kg sc) on plasma cytokine concentrations at 16 h after a Tur injection. Data points represent means ⫾ SD (n ⫽ 4). a Significantly different from control mice (time, 0 h), p ⬍ 0.05. b Significantly different from Tur-injected mice, p ⬍ 0.05.
caused significant increases in plasma IL-6 and pretreatment of mice with Dex suppresses this elevation. DISCUSSION
In the present experiments we demonstrate clear evidence that pretreatment with a glucocorticoid significantly suppresses the inflammation-mediated tolerance to Cd toxicity, and the suppression was seen in regard to both overall lethality and hepatotoxicity but not in testicular toxicity. Both the inflammation-induced tolerance to Cd and the suppression of this tolerance by glucocorticoid appear to involve induction of hepatic MT. It has been demonstrated that glucocorticoid directly induces hepatic MT synthesis via GRE (Kelly et al., 1997) and glucocorticoid acts synergistically with IL-6 in the transcriptional activation of the mouse MT-I gene (Itoh et al., 1996) and in induction of hepatic MT (Sato et al., 1996). Although injection of Dex to control mice increased hepatic MT concentrations twofold, hepatic MT concentrations in the Dex⫹Tur-mice were decreased by two-thirds relative to Tur-mice and were approximately four times higher than levels in normal control mice. Moreover, marked elevations in plasma IL-6 concentration after Tur-initiated inflammation were suppressed by pretreatment with Dex. It has been reported that IL-6 directly induces MT synthesis and mediates induction of acute phase proteins, such as MT, in inflammation responses initiated by LPS, Tur, and n-hexane (Min et al., 1991). Therefore, the
conflicting effects shown here of Dex on hepatic MT induction suggest that Dex both may enhance hepatic MT induction by IL-6 and may repress cytokine gene transcription in inflammatory cells. Since MT induced by the inflammatory response reduces access of Cd to molecular targets in the hepatocyte, the dimunition of Cd-induced lethality and hepatic injury is likely dependent on hepatic MT levels. Inflammation also protects against acute Cd-induced extrahepatic toxicity. The induction of hepatic MT by other compounds involved in Cd responses results in a decreased amount of Cd reaching the testes (Shiraishi et al., 1993, 1994; Sauer et al., 1997a). In fact, pretreatment with Tur resulted in significant decreases in renal Cd retention with a concomitant increase in hepatic Cd retention. Thus, hepatic MT induction following inflammation both protects against liver injury and may result in a change in the distribution of Cd. Conversely, suppression of MT induction by pretreatment with glucocorticoid (Dex⫹Tur-mice) results in a reoccurrence of Cd hepatotoxicity. Hepatic MT concentrations in the Dex⫹Tur-mice were four times higher than in control mice and two times higher than in Dex-alone-treated mice. Therefore, hepatic MT levels in the Dex⫹Tur-mice may be high enough to protect against testicular Cd toxicity or to affect the distribution of Cd in the testes. Administration of pharmacological doses of glucocorticoid appears to increase the endogenous release of antiinflammatory cytokine IL-10 in humans and mice (Marchant et al., 1996; Van der Poll et al., 1996). Thus, pretreatment with a glucocorticoid alone may prevent Cd toxicity not only due to an increase in hepatic MT induction but also due to an enhancement in the production of the antiinflammatory cytokines. However, as we have shown, pretreatment of Tur-mice with glucocorticoid (Dex⫹Tur-mice) suppressed the inflammationmediated tolerance to Cd. We suggest that glucocorticoid might preferentially suppress hepatic MT induction over the cytokine protective effects. In summary, it appears that pretreatment with glucocorticoid significantly suppresses the tolerance of inflammation resulting from Cd toxicity through suppression of hepatic MT synthesis. This suppression in MT synthesis was caused by decreases in inflammatory cytokine production, such as IL-6. Glucocorticoids appear to affect hepatic MT induction mediated via inflammatory cytokines preferentially rather than through direct induction. We suggest that the inflammatory response modulates Cd toxicity through induction of MT by cytokines. Pretreatment with glucocorticoid suppresses hepatic MT induction by cytokines. Hepatic MT induction may be involved not only in protecting against liver injury but also by altering the distribution of Cd in extrahepatic organs. It is well known that MT-I/II knockout mice are more sensitive to Cd toxicity than normal mice (Klaassen and Liu, 1998). Future investigations employing tissue-specific expression of MT genes will help to elucidate the specific toxicological roles of hepatic MT in mediating Cd toxicity.
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Glucocorticoids Suppress the Inflammation-Mediated Tolerance to Acute Toxicity of Cadmium in Mice Kyong-Son Min, 1 Heisuk Kim, Miho Fujii, Noriko Tetsuchikawahara, and Satomi Onosaka Department of Nutrition, Kobe Gakuin University, Ikawadani-cho, Nishi-ku, Kobe, 651-2180, Japan Received April 13, 2001; accepted October 15, 2001
parenterally at high doses, Cd 2⫹ can induce lethal hepatocellular necrosis as well as extensive testicular lesions (Sauer et al., 1997a; Rikans and Yamano, 2000). It has been demonstrated that Cd 2⫹ induces multiple cytotoxic and/or metabolic effects, such as interference with the action of essential metals (Chmielnicka and Sowa, 1996) and alteration of the activity of various enzymes (Kinne-Saffran et al., 1993; Casalino et al., 1997; Patra et al., 1999). Hepatic injury due to Cd exposure can involve both direct and indirect damage to the hepatocyte, as seen with some other xenobiotics. Increased expression of several cytotoxic mediators, including tumor necrosis factor (TNF)-␣, has been demonstrated in rodent liver or hepatocytes following acute Cd 2⫹ exposure (Kayama et al., 1995a,b; Dong et al., 1998). It has been demonstrated that Kupffer cell inhibitors or enhancers modulate the levels of hepatotoxicity induced by certain xenobiotics (Edwards et al., 1993; El Sisi et al., 1993; Blazka et al., 1995; Michael et al., 1999). However, either its inhibitors or enhancers resulted in the attenuation of Cd-induced hepatotoxicity (Sauer et al., 1997a,b). The mechanism of the tolerance to Cd is thought to be mediated by the induction of hepatic metallothionein (MT). These data suggest that cytokines might demonstrate a preference for the protective effect by hepatic MT induction over the cytotoxic effects on progression of Cd-induced liver injury. Therefore, sequestration of Cd by MT in the early stages, post-Cd dosing, is the most effective mechanism for decreasing Cd toxicity. Several compounds, including metals, steroid hormones, calmodulin inhibitors, antioxidants, and inflammatory agents have been shown to promote tolerance to Cd-induced injury by inducing synthesis of MT, either directly or indirectly (Goering and Klaassen, 1984a,b; Shiraishi et al., 1993, 1994; Sauer et al., 1997b). The proposed mechanism of this MT-mediated Cd-tolerance involves sequestering Cd 2⫹ from molecular targets by high-affinity binding of MT to Cd 2⫹. MT is a small, cysteine-rich, metal-binding protein that plays a role in zinc homeostasis, heavy metal detoxification, and the protection of cells against reactive oxygen intermediates and electrophilic anticancer drugs. The rapid induction of MT gene transcription by heavy metals is mediated through metal response elements, which are present in multiple copies in the proximal promoters
Glucocorticoids Suppress the Inflammation-Mediated Tolerance to Acute Toxicity of Cadmium in Mice. Min, K.-S., Kim, H., Fujii, M., Tetsuchikawahara, N., and Onosaka, S. (2002). Toxicol. Appl. Pharmacol. 178, 1–7. Several compounds have been shown to cause acute toxicity to cadmium (Cd). The mechanism of tolerance to Cd toxicity induced by glucocorticoids or by inflammation involves induction of metallothionein (MT) synthesis via glucocorticoid response elements or by inflammatory cytokines. We have demonstrated previously that the synthetic glucocorticoid dexamethasone suppresses inflammation-mediated induction of hepatic MT synthesis. Here we investigated the effect of glucocorticoid on tolerance to Cd induced by inflammation in mice. The LD50 of Cd for mice with induced inflammation by injection with turpentine oil (Turmice) was higher than the LD50 in control mice. Pretreatment of Tur-mice with dexamethasone to the Tur-mice (DexⴙTur-mice) resulted in a decrease in LD50 after Cd treatment. A significant increase in plasma alanine aminotransferase and aspartate aminotransferase levels in the DexⴙTur-mice was observed at lower doses of Cd than in the Tur-mice and at higher doses of Cd than in control mice. Dexamethasone did not suppress tolerance to cadmium toxicity in the testes of the Tur-mice. Pretreatment of Tur-mice with dexamethasone resulted in suppression of both plasma interleukin (IL)-6 elevation and in suppression of hepatic MT levels when induced by inflammation but not when induced by Cd. These data suggest that suppression of tolerance to Cd toxicity induced by glucocorticoid may involve hepatic MT synthesis mediated by inflammatory cytokines, such as IL-6. We suggest that the inflammatory response can modulate Cd toxicity by induction of MT by inflammatory cytokines. © 2002 Elsevier Science Key Words: cadmium; inflammation; glucocorticoid; inflammatory cytokine; tolerance.
Cadmium (Cd) is a widespread environmental pollutant that poses a significant health risk to humans and animals. It has been found that Cd 2⫹ can produce both acute and chronic tissue injury and can damage various organs including the lung, liver, kidney, bone, testis, and placenta, depending on the dose, route, and duration of exposure (for reviews, see Goering et al., 1995; Jarup et al., 1998). For example, when administered 1
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of the MT genes. MT-I and MT-II are acute phase proteins induced in response to a variety of physiological and pharmacological agents such as glucocorticoids, inflammatory compounds, and interleukins. Glucocorticoid directly induces MT synthesis via the glucocorticoid response elements (GRE). Induction of MT synthesis by inflammation following injection of turpentine oil, n-hexane, or lipopolysaccharide (LPS) has been shown to be mediated through inflammatory cytokines (De et al., 1990; Min et al., 1991; Itoh et al., 1994, 1996). We have shown previously that glucocorticoid directly increases hepatic MT synthesis in mice and can also suppress inflammation-induced increases in MT (Min et al., 1992). In contrast, in both animal and cell culture studies, dexamethasone induced MT synthesis synergistically with increases in IL-6 (Sato et al., 1996; Kasutani et al., 1998). We suggest that glucocorticoid may both inhibit the production of cytokines and may stimulate cytokine-mediated induction of MT, resulting in glucocorticoid having two opposing functions in the induction of hepatic MT. Moreover, inflammatory cytokines are involved in Cd hepatotoxicity. Here we have investigated the effects of glucocorticoids on inflammation-induced Cd toxicity in mice.
FIG. 1. Effect of pretreatment with Tur and/or Dex on acute lethal toxicity of Cd at various doses. CdCl 2 (0.2–3.0 mg Cd/kg) was injected to pretreated mice (0 – 0.6 mg Cd/kg; 12 mice/each dose, 0.8 –3.0 mg Cd/kg; 16 mice/each dose) with saline, turpentine oil (Tur, 10 ml/kg sc), and/or dexamethasone (Dex, 25 mg/kg sc). Survived number was counted after 48 h. Control, nonpretreated mice; Tur, Tur-pretreated mice; Dex⫹Tur, pretreatment with Tur to Dex-pretreated mice.
MATERIALS AND METHODS Animals. Male mice (ddY strain) 6 weeks old and weighing approximately 30 g were purchased from Nihon SLC (Shizuoka, Japan). Mice were maintained on a 12-h light– dark cycle and given food and water ad libitum (Type MF, Oriental Yeast, Osaka, Japan). All animal experiments were carried out under the control of the Animal Research Committee, in accordance with the Guidelines on Animal Experiments in Kobe Gakuin University and Japanese Government Animal Protection and Management Law (No. 105). Chemicals. All chemicals were purchased from Nakarai Tesque, Inc. (Kyoto, Japan). Pretreatment of mice with turpentine oil and/or dexamethasone. Two hundred milligrams of dexamethasone (Dex), a synthetic glucocorticoid, was suspended in 10 ml of 5% acacia (Arabic gum) solution. Typically, 24 h following pretreatment with Dex (25 mg/kg) or its vehicle, the mice were treated subcutaneously with 10 ml/kg turpentine oil (Tur) or saline. After an additional 24 h, the animals received a single dose of CdCl 2 (0.2–3.0 mg Cd/kg iv) in 10 ml/kg saline via the femoral vein and either were observed or were euthanized by overanesthesia at the indicated time. Heparinized blood was collected from the posterior vena cava 16 h after injection of Cd. Sections of liver and kidney were homogenized with 0.25 M sucrose solution and then were analyzed for both Cd and MT concentrations. Testes were taken 48 h post-Cd injection and were homogenized with Tris buffer (10 mM, pH 7.6) for analysis of testicular hemoglobin concentrations. Plasma activities of hepatic enzymes. Plasma activities of both alanine aminotransferase (ALT) and aspartate aminotransferase (AST) were determined spectrophotometrically using Transaminase CII Wako kits (Wako Pure Chemical Industries, Ltd., Osaka, Japan). Absorbance of hemoglobin. To quantify the extent of cadmium-induced testicular hemorrhage, hemoglobin levels in the 18,000g supernatant fraction from testicular homogenates were determined by measuring absorbance at 414 nm by the method of Niewenhuis and Prozialeck (1987). Analysis of Cd concentration. Following overnight digestion of the tissue homogenates with nitric acid, the solutions were brought to a final volume of 4 ml with water and were filtered using a 0.45-m Dismic-13CP syringe tip
filter. Samples were analyzed for Cd 2⫹ concentration using a flame atomic absorption spectrophotometer (Z-5000 Polarized Zeeman Atomic Absorption Spectrophotometer, Hitachi, Tokyo, Japan). Determination of MT. Hepatic MT concentrations were determined by the Cd– heme method as described previously (Onosaka et al., 1978). Measurement of plasma IL-6 and TNF-␣. The concentrations of interleukin-6 (IL-6) and tumor necrosis factor-␣ (TNF-␣) in plasma were measured by ELISA. Purified rat anti-mouse IL-6 and the TNF-␣ monoclonal antibodies were obtained from Fujisawa Pharmaceutical Co. Ltd. (Pharmigen, San Diego, CA). Recombinant mouse IL-6 and TNF-␣ obtained from Fujisawa Pharmaceutical Co. Ltd. (Pharmigen) were used as standards. Statistics. Results are expressed as means ⫾ SD. Data were analyzed for significance by Student’s t test and by a two-tailed ANOVA. Differences were considered significant at p ⬍ 0.05.
RESULTS
Effect of Pretreatment with Tur and/or Dex on the Lethal Toxicity of Cd The Tur and/or Dex-pretreated mice were observed 24 h following administration of increasing doses of Cd (Fig. 1) and survival rates (%) were calculated. The LD50 of Cd was 1.25 mg Cd/kg for control mice. When mice were given an injection of turpentine oil to initiate inflammation (Turmice), the LD50 for Cd increased to 2.5 mg Cd/kg. However, in mice pretreated with Dex 24 h prior to injection with Tur (Dex⫹Tur-mice), the LD50 for Cd was 2 mg Cd/kg, lower than in the Tur-treated mice but higher than in control mice. These data suggest that initiation of inflammation
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FIG. 2. Effect of pretreatment with Tur and/or Dex on Cd hepatotoxicity. Activities of aminotransferases (ALT and AST) were determined at 16 h after an injection of Cd. Data points represent means ⫾ SD (n ⫽ 4–8). *Significantly different from no Cd-injected mice (0 mg Cd/kg), p ⬍ 0.05.
resulted in a tolerance to Cd toxicity and that glucocorticoid suppresses this tolerance. Effect of Pretreatment with Tur and/or Dex on Cd-Induced Hepatotoxicity and Testicular Toxicity Mice administered CdCl 2 demonstrated clear signs of liver injury, as measured by plasma levels of ALT and AST 16 h postdosing (Fig. 2). Pretreatment of the mice with Tur (Turmice) and/or Dex (Dex⫹Tur-mice) significantly attenuated the observed hepatotoxicity at a Cd dose of 1 mg/kg. Significant increases in plasma transaminase activities in the Dex⫹Turmice were observed at higher doses of Cd than in control mice and at lower doses of Cd than in Tur-treated mice. After a Cd injection at a dose of 1.5 mg/kg, no overt morphological alterations were associated with Cd in Tur-mice, but the hepatocellular necrosis was observed in Dex⫹Tur-mice (data not shown). Cd injection resulted in an increase in testicular hemoglobin after 48 h (Fig. 3), an indication of Cd-induced testicular hemorrhage. A significant increase in testicular hemoglobin concentration occurred at 1 mg Cd/kg in control mice. Testicular hemoglobin concentrations were significantly elevated in the Tur-mice and Dex⫹Tur-mice at Cd doses greater than 1.5 mg Cd/kg. Therefore, turpentine-induced inflammation seems to reduce the sensitivity of mice to acute Cd effects, such as hepatotoxicity and testicular toxicity, and glucocorticoid can decrease the inflammation-mediated tolerance to hepatotoxicity but does not affect testicular toxicity.
FIG. 3. Effect of pretreatment with Tur and/or Dex on testicular Cd toxicity. Testicular hemoglobin concentrations were measured at 48 h after an injection of CdCl 2 . Data points present means ⫾ SD of five mice. *Significantly different from no Cd-injected mice (0 mg Cd/kg), p ⬍ 0.05.
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FIG. 4. Effect of pretreatment with Tur and/or Dex on hepatic and renal Cd concentration. The concentrations of Cd were determined after 16 h. Data points represent means ⫾ SD (n ⫽ 4–8). a Significantly different from no Cd-injected mice, p ⬍ 0.05. b Significantly different from Tur-injected mice, p ⬍ 0.05.
Effect of Pretreatment with Tur and/or Dex on Hepatic and Renal Cd Retention Cd concentrations were measured in liver and kidney, tissues that typically retain Cd, 16 h following an injection of Cd (Fig. 4). Hepatic Cd accumulation following the dose of 1 mg Cd/kg in both Tur-mice and Dex⫹Tur-mice was higher than in the nonpretreated control mice. No significant difference in hepatic Cd accumulation between the Tur-mice and the Dex⫹Tur-mice was observed except at the 1–1.25 mg Cd/kg doses. In contrast, renal accumulation of Cd was significantly reduced in the Tur-mice compared to control mice at 1 mg Cd/kg. In addition, renal Cd accumulation was higher in Dex⫹Tur-mice than in the Tur-mice. The contrast in hepatic and renal distributions indicates that a reduction of Cd accumulation by extrahepatic tissues, such as in the kidneys, is offset by increased Cd accumulation by the liver. Effect of Pretreatment with Tur and/or Dex on Hepatic MT Concentration before or after Cd Injection In the liver samples used for analysis of Cd levels, hepatic MT concentrations 16 h following Cd injection were determined and are shown in Fig. 5. In agreement with a previous report from our laboratory (Min et al., 1992), treatment with Tur caused a significant increase in hepatic MT over control and this effect was suppressed by pretreatment of the mice with Dex before an injection of Cd. Hepatic MT concentrations in the Tur-treated mice significantly increased at all doses of Cd examined. Pretreatment of the Tur-mice with Dex resulted in a
FIG. 5. Effect of pretreatment with Tur and/or Dex on hepatic MT concentration after an injection of Cd. Hepatic MT concentrations were determined at 16 h after an injection of Cd. a Significantly different from no Cd-injected mice, p ⬍ 0.05. b Significantly different from Tur-injected mice, p ⬍ 0.05.
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FIG. 6. Effect of preinduced MT by Dex at various doses on hepatotoxicity of Cd. (A) Effect of preinduced MT by Dex (0 –100 mg/kg sc) without Tur on hepatotoxicity of Cd (1 mg Cd/kg sc). (B) Effect of preinduced MT by Dex (0 –100 mg/kg sc) with Tur on hepatotoxicity of Cd (1.5 mg Cd/kg sc). Hepatic MT concentrations were determined before an injection of Cd. Plasma ALT activities and testicular hemoglobin concentrations were determined at 16 and 48 h after a subsequent Cd injection, respectively. Data points represent means ⫾ SD (n ⫽ 4). *Significantly different from Dex (0 mg/kg) mice, p ⬍ 0.05.
slight suppression of these increases in hepatic MT. Combined, these results indicate that glucocorticoid can suppress hepatic induction of MT that results from Tur-initiated inflammation, but glucocorticoid does not alter subsequent Cd-induced changes in MT levels. Effect of MT Preinduction by Dex with or without Tur on Cd Toxicity Treatment with Dex alone resulted in a significant increase of hepatic MT concentrations independent of dose and caused dimunition of plasma ALT elevation following injection of Cd at a dose of 1 mg/kg (Fig. 6A). An injection of Dex alone did not affect plasma ALT activity before Cd injection (data not shown). The effects of the pretreated dose of Dex in Tur-mice on hepatic MT concentration before an injection of Cd, plasma ALT activity, and testicular hemoglobin concentrations are shown in Fig. 6B. Treatment with Tur alone (Dex 0 mg/kg) resulted in a remarkable increase of hepatic MT concentrations and diminished plasma ALT elevation following injection of Cd at a dose of 1.5 mg/kg (Fig. 6B). In contrast with pretreat-
ment of control mice with Dex (Fig. 6A), pretreatment of Tur-mice with Dex resulted in significant decreases of hepatic MT concentrations at a dose of 25–100 mg/kg. According to the significant decrease of hepatic MT concentration, pretreatment of the Tur-mice with Dex did not diminish Cd-induced ALT elevation. Finally, Dex pretreatment did not relapse Cdinduced testicular toxicity in these mice, as seen by the lack of changes in testicular Hb levels. Effect of Pretreatment with Dex on Plasma Concentration of IL-6 and TNF-␣ after an Injection of Tur IL-6 and TNF-␣ levels were determined following injection of mice with Tur, in the absence or presence of Dex pretreatment (Fig. 7). There was a marked elevation in plasma IL-6 level 16 h following injection with Tur. Although both Dex and Tur treatments alone resulted in significant increases in plasma IL-6 concentrations, pretreatment with Dex to Tur-mice resulted in IL-6 levels that were similar to those seen with Dex only. Tur treatment did not result in elevated plasma TNF-␣. These results indicate that the Tur-initiated inflammation
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FIG. 7. Effect of Dex on plasma concentration of cytokines following inflammation initiated by Tur. (A) Plasma concentration of IL-6 and TNF following inflammation initiated by Tur (10 ml/kg sc). (B) Effect of pretreatment with Dex (25 mg/kg sc) on plasma cytokine concentrations at 16 h after a Tur injection. Data points represent means ⫾ SD (n ⫽ 4). a Significantly different from control mice (time, 0 h), p ⬍ 0.05. b Significantly different from Tur-injected mice, p ⬍ 0.05.
caused significant increases in plasma IL-6 and pretreatment of mice with Dex suppresses this elevation. DISCUSSION
In the present experiments we demonstrate clear evidence that pretreatment with a glucocorticoid significantly suppresses the inflammation-mediated tolerance to Cd toxicity, and the suppression was seen in regard to both overall lethality and hepatotoxicity but not in testicular toxicity. Both the inflammation-induced tolerance to Cd and the suppression of this tolerance by glucocorticoid appear to involve induction of hepatic MT. It has been demonstrated that glucocorticoid directly induces hepatic MT synthesis via GRE (Kelly et al., 1997) and glucocorticoid acts synergistically with IL-6 in the transcriptional activation of the mouse MT-I gene (Itoh et al., 1996) and in induction of hepatic MT (Sato et al., 1996). Although injection of Dex to control mice increased hepatic MT concentrations twofold, hepatic MT concentrations in the Dex⫹Tur-mice were decreased by two-thirds relative to Tur-mice and were approximately four times higher than levels in normal control mice. Moreover, marked elevations in plasma IL-6 concentration after Tur-initiated inflammation were suppressed by pretreatment with Dex. It has been reported that IL-6 directly induces MT synthesis and mediates induction of acute phase proteins, such as MT, in inflammation responses initiated by LPS, Tur, and n-hexane (Min et al., 1991). Therefore, the
conflicting effects shown here of Dex on hepatic MT induction suggest that Dex both may enhance hepatic MT induction by IL-6 and may repress cytokine gene transcription in inflammatory cells. Since MT induced by the inflammatory response reduces access of Cd to molecular targets in the hepatocyte, the dimunition of Cd-induced lethality and hepatic injury is likely dependent on hepatic MT levels. Inflammation also protects against acute Cd-induced extrahepatic toxicity. The induction of hepatic MT by other compounds involved in Cd responses results in a decreased amount of Cd reaching the testes (Shiraishi et al., 1993, 1994; Sauer et al., 1997a). In fact, pretreatment with Tur resulted in significant decreases in renal Cd retention with a concomitant increase in hepatic Cd retention. Thus, hepatic MT induction following inflammation both protects against liver injury and may result in a change in the distribution of Cd. Conversely, suppression of MT induction by pretreatment with glucocorticoid (Dex⫹Tur-mice) results in a reoccurrence of Cd hepatotoxicity. Hepatic MT concentrations in the Dex⫹Tur-mice were four times higher than in control mice and two times higher than in Dex-alone-treated mice. Therefore, hepatic MT levels in the Dex⫹Tur-mice may be high enough to protect against testicular Cd toxicity or to affect the distribution of Cd in the testes. Administration of pharmacological doses of glucocorticoid appears to increase the endogenous release of antiinflammatory cytokine IL-10 in humans and mice (Marchant et al., 1996; Van der Poll et al., 1996). Thus, pretreatment with a glucocorticoid alone may prevent Cd toxicity not only due to an increase in hepatic MT induction but also due to an enhancement in the production of the antiinflammatory cytokines. However, as we have shown, pretreatment of Tur-mice with glucocorticoid (Dex⫹Tur-mice) suppressed the inflammationmediated tolerance to Cd. We suggest that glucocorticoid might preferentially suppress hepatic MT induction over the cytokine protective effects. In summary, it appears that pretreatment with glucocorticoid significantly suppresses the tolerance of inflammation resulting from Cd toxicity through suppression of hepatic MT synthesis. This suppression in MT synthesis was caused by decreases in inflammatory cytokine production, such as IL-6. Glucocorticoids appear to affect hepatic MT induction mediated via inflammatory cytokines preferentially rather than through direct induction. We suggest that the inflammatory response modulates Cd toxicity through induction of MT by cytokines. Pretreatment with glucocorticoid suppresses hepatic MT induction by cytokines. Hepatic MT induction may be involved not only in protecting against liver injury but also by altering the distribution of Cd in extrahepatic organs. It is well known that MT-I/II knockout mice are more sensitive to Cd toxicity than normal mice (Klaassen and Liu, 1998). Future investigations employing tissue-specific expression of MT genes will help to elucidate the specific toxicological roles of hepatic MT in mediating Cd toxicity.
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