Acute-phase protein expression in DMSO-intoxicated rats

Toxicology Letters 147 (2004) 153–159

Acute-phase protein expression in DMSO-intoxicated rats Svetlana Ivanovi´c Mati´c, Svetlana Dini´c, Mirjana Mihailovi´c, Ilijana Grigorov, Desanka Bogojevi´c, Goran Poznanovi´c∗ Molecular Biology Laboratory, Institute for Biological Research, 29th November, 11060 Belgrade, Serbia and Montenegro Received 18 August 2003; received in revised form 30 October 2003; accepted 4 November 2003

Abstract The ability of dimethyl sulfoxide (DMSO) to induce the acute-phase (AP) response was examined. Injection of DMSO to laboratory rats caused a rapid doubling of the plasma corticosterone concentration 2 h after treatment. The elevated corticosterone concentration promoted the synthesis of mRNAs for several AP reactants. At 24 h after DMSO administration the relative serum concentration of cysteine-proteinase inhibitor (CPI) increased about 710%, ␣1 -acid glycoprotein (AGP) 630%, ␣1 -macroglobulin (MG) 510%, ␥ fibrinogen (Fb) 420%, haptoglobin (Hp) 280%, whereas the relative concentration of albumin, a “negative” AP reactant, decreased to 93%. The extent and kinetics of the corticosterone increase and the general increase of AP reactant mRNAs and protein serum concentrations after DMSO administration corresponded to the overall changes observed during the turpentine-induced AP response. On the basis of these findings it was concluded that DMSO was capable of promoting the AP response in rats. © 2003 Elsevier Ireland Ltd. All rights reserved. Keywords: Acute-phase reactants; Corticosterone; DMSO; Inflammation

1. Introduction A variety of stressors: infections (Koj et al., 1982; Rothenburger et al., 1999), physical trauma such as thermal (Ševaljevi´c et al., 1988, 1989a,b) or chemical injury (Glibeti´c et al., 1992; Ivanovi´c-Mati´c and Poznanovi´c, 1996) and ionizing radiation (Magi´c et al., 1995) trigger a homeostatic process known as inflammation that is aimed at restoring an organism’s normal functioning. The activation of tissue macrophages or blood monocytes leads to the ∗ Corresponding author. Tel.: +381-11-764-422; fax: +381-11-761-433. E-mail address: [email protected] (G. Poznanovi´c).

production of soluble mediators of inflammation that ultimately serve to mobilize the metabolic response of the whole organism (Baumann and Gauldie, 1994; Gabay and Kushner, 1999; Koj, 1998). During the early phase of inflammation, the immediate set of induced reactions is refered to as the acute-phase (AP) response. The cytokines (IL-6, IL-1␤, TNF-␣) that are produced are the major inducers of fever and neuroendocrine changes; most notably they activate the paraventricular–infundibular corticotropin-releasing hormone system. This stress system stimulates ACTHproducing cells in the pituitary gland. The realeased ACTH in turn simulates the adrenal glands to secrete glucocorticoid hormones. Glucocorticoids modulate energy metabolism, the immune system and promote

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the synthesis of the so-called AP proteins or reactants in the liver (Richards et al., 1991; Moshage, 1997; Streetz et al., 2001). The AP proteins have a variety of activities, from those contributing to the host’s defense either by directly neutralizing inflammatory agents, helping to minimize the extent of local tissue damage, to those participating in tissue repair and regeneration. Proteinase inhibitors, coagulation proteins, complement factors, transporters or scavengers belong to the group of AP proteins (Ruminy et al., 2001). Although the plasma concentration of many AP proteins increases, it does not increase uniformly under different stress conditions (Poznanovi´c et al., 1997; Gabay and Kushner, 1999). The observed subtle variations suggest that AP proteins are individually regulated by the differences in the patterns and levels of production of specific cytokines and other inflammatory mediators (Engler, 1995). In contrast to the AP reactants, the concentrations of plasma albumin, transferrin and ␣2 -HS glycoprotein fall during the AP response and the term “negative AP reactants” has been ascribed to these proteins (Kushner, 1982). Dimethyl sulfoxide (DMSO) or (CH3 )2 SO is an amphipathic molecule with a polar domain and two apolar groups that contribute towards its solubility in both aqueous and organic media (Santos et al., 2003). DMSO is often used for insecticide solvation and in laboratory research as a cryopreservative for cultured cells (Song et al., 1995; Jacob and Herschler, 1986). In laboratory studies a variety of primary pharmacological actions of DMSO have been documented, such as effects on membrane transport and connective tissue, anti-inflammatory activity, nerve blockade (analgesia), bacteriostasis, diuresis, enhancement and/or reduction of the effectiveness of other drugs, cholinesterase inhibition, non-specific enhancement of resistance to infection, vasodilation, muscle relaxation, antagonism to platelet aggregation and influence on serum cholesterol in experimental hypercholesterolemia (Jacob and Herschler, 1986; Evans et al., 1993; Rodriguez-Burford et al., 2003). It has been reported that DMSO induces differentiation and functioning of leukemic and other malignant cells, has prophylactic radioprotective properties and cryoprotective actions, and that it protects against ischemic injury (Jacob and Herschler, 1986). The rapid penetration of DMSO and its enhancing effect on the uptake of other substances

across biological membranes, free radical scavenging (Gonskyi et al., 1991), effects on coagulation and anti-cholinesterase activity (Schreiber and Slapke, 1990; Pestronk et al., 1985; Pestronk and Drachman, 1980; Santos et al., 2003) are some of the properties of DMSO deemed to be important for its therapeutic as well as toxic actions. We have previously described the effects of organophosphorus cholinesterase inhibitors, soman and paraoxone (Ševaljevi´c et al., 1989c, 1990, 1992; Ivanovi´c-Mati´c et al., 1999), on AP protein expression in rat liver. In light of the reported anti-cholinesterase activity of DMSO (Schreiber and Slapke, 1990; Pestronk et al., 1985; Gupta et al., 2000) the ability of DMSO to induce the AP response could also be considered. However, while some studies suggest a relationship between DMSO and the AP reaction (Iwasa et al., 1988) others do not (Brayton, 1986; Jacob and Herschler, 1986). Such conflicting data led us to examine the ability of DMSO to induce the AP response. To that end, the time course of changes in the serum levels of corticosterone and select AP proteins after DMSO administration were examined. Such studies are important in light of the many uses of DMSO.

2. Materials and methods 2.1. Materials DMSO and corticosterone were obtained from the Sigma Chemical Co. (St. Louis, MO, USA), turpentine was obtained from Kemika (Zagreb, Croatia), Freund’s adjuvant was obtained from the Torlak Institute for Vaccines and Serums (Belgrade, Serbia and Montenegro), nitrocellulose filters (BA 85; 0.45 um pore size) were obtained from Schleicher and Schuell (Keene, NH, USA), deoxycitidine 5 -[␣-32 P] triphosphate (specific activity 9.25 Mbq/mmol) and the nick translation kit were obtained from Amersham International Plc. (Amersham, Buckinghamshire, UK). pBR322 containing cDNA inserts at the PstI site and encoding for the following rat plasma proteins, were obtained from Dr. H. Baumann (Roswell Park Memorial Institute, Buffalo, NY, USA): ␣1 -acid glycoprotein (AGP), haptoglobin (Hp), cysteine-proteinase inhibitor (CPI), ␥-fibrinogen (Fb), for ␣2 -macroglobulin (MG) from Dr. P. Heinrich

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(Institut fur Biochemie der RWTH Aachen, Germany), and for albumin (Al) from Dr. T.D. Sargent (California Institute of Technology, Pasadena, CA, USA). All of the chemicals were reagent grade. 2.2. Animals Ten weeks old male Wistar rats weighing between 200 g and 300 g were used. The animals were divided into three groups according to the administered agents. One group of rats received interperitoneal injections (1 mg/g body weight) of DMSO. The second group was given subcutaneously (1 ␮l/g body weight) turpentine, the oleoresin from Pinus pinaceae sp., a commonly used inducer of the AP response in laboratory animals (Baumann et al., 1984). The control group received saline. The rats were kept at constant temperature, humidity and dark/light intervals and killed at the indicated times.

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Laboratories, Bethesda, MD, USA), and hybridised with plasmids labelled to a specific activity of about 107 dpm/␮g DNA by nick translation. The radioactivity used for the hybridization was not lower than 3 × 106 dpm/ml hybridizing solution. The filters were exposed to Kodak-X-omat film. Relative changes in mRNA concentration were determined by scanning the autoradiograms and expressing as mean percentages relative to the control (100%) using a PhosphorImagerTM (Molecular Dynamics) and ImageQuant ver. 3.3 software (a standard curve was drawn for every probed plasmid from the hybridization data obtained from the three different amounts of control RNA). 2.5. Anti-serum preparation and crossed immunoelectrophoresis

Blood samples for corticosterone determination were taken from the abdominal aorta of ether anaesthetized rats at 14:00 h. The concentration was determined as described (Hodges and Sadow, 1967). Corticosterone was extracted from the plasma (200 ␮l were diluted to 2 ml with distilled H2 O) with 7.0 ml of chloroform. Following the removal of the aqueous layer, the chloroform was extracted with 1 M NaOH, followed by water. Two millilitres of H2 SO4 in 50% ethanol (2.5:1 v/v) were added to the chloroform and after 2 h the fluorescence of the acid layer was measured against an acid–alcohol blank at 470 nm (excitation wavelength) and 520 nm (emission) using a spectrophotofluorimeter (Amico-Bowman). The corticosterone concentration was expressed in ␮g/100 ml plasma.

The serum from turpentine-treated rats was used for the preparation of antibodies against the AP proteins. Rats were killed 24 h after the induction of the AP reaction by turpentine injection. The sera (150 ␮l) were diluted with saline (350 ␮l), mixed with complete Freund’s adjuvant (1:1) and subcutaneously injected into rabbits. Over a period of 2 weeks, three to five more injections were given with incomplete Freund’s adjuvant. Crossed immunoelectrophoresis of rat serum proteins was performed according to the described procedure (Weeke, 1970; Laurell, 1972). After the optimal antigen–antibody concentration ratios were determined, the established (same) polyspecific antigen concentration was used throughout in all of the subsequent experiments. The areas under the obtained immunoprecipitation peaks were quantified and the relative percent increase in concentration of AGP, CPI, Hp and MG expressed relative to the respective control peak area that was assumed to be 100% (Ševaljevi´c et al., 1989a, 1989b, 1990). The gels were stained with Coomassie Blue.

2.4. Dot-blot analysis

2.6. Determination of Al and Fb concentrations

Total rat liver RNA was isolated according the procedure based on the extractions of RNA with guanidine–HCl described by Cox (1986). RNA samples were dot-blotted in triplicate (2.5 ␮g/spot, 5.0 ␮g/spot and 10 ␮g/spot) onto nitrocellulose filters using a Hybri Dot Manifold (Bethesda Research

Al was determined by the method of Lowry et al. (1951) after the precipitation of globulins with polyethylene glycol (Gamball, 1971). Fb was determined as described by Koj and McFarlane (1968). The changes in their concentrations were expressed as percentages with respect to the control values (100%).

2.3. Determination of corticosterone concentrations

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3. Results and discussion The increase in concentration of circulating AP proteins is a marked feature of the systemic stress reaction (Ševaljevi´c et al., 1989a, 1989b, 1989c). In the present paper we examined whether DMSO administration could bring about an early increase of corticosterone concentration in the blood and the subsequent increased synthesis and rise in circulating AP protein concentrations. A comparison of the temporal changes of serum corticosterone concentrations after treatment with DMSO and turpentine, the oft-used inducer of the AP response in laboratory animals, is shown in Fig. 1. Corticosterone peaked 2 h after the administration of either agent. The injection of turpentine and DMSO, respectively, led to about 320% and 300% increases of corticosterone concentrations. Four hours after treatment the corticosterone concentrations remained 280% and 260% higher in the turpentine- and DMSO-treated rats, respectively. By the 12th hour after administration the corticosterone concentrations approached the basal values in both groups of treated rats. We concluded that DMSO exerted the same effect on corticosterone induction as turpentine. Corticosterone either directly stimulates or, by enhancing the effects of interleukin-6 and -1 type cytokines, acts synergistically on the expression of AP protein genes (Baumann and Gauldie, 1994; Scotte

Fig. 1. Change of serum corticosterone concentrations after turpentine and DMSO administration. Rats were divided into groups according to the administered agents: one group received turpentine (␮l/g s.c.), the second group DMSO (mg/g s.c.), and the control group saline. The values are mean ± S.E. from three separate experiments.

et al., 1996; Wiegers and Reul, 1998). The relative changes in the concentrations of several major AP protein mRNAs 12 h after turpentine and DMSO injections were monitored by dot-blot analysis (Fig. 2). It should be noted that in the course of the AP response the maximal increase in transcriptional activity of AP protein genes is established 12 h after induction (Shiels et al., 1987; Ševaljevi´c et al., 1989b; Adler et al., 2001). This is prior to the establishment of the maximal, plateau values of AP protein concentrations in the circulation that are observed between 24 h and 48 h after induction of the AP response (Koj, 1974; Kushner, 1982; Ruminy et al., 2001). At 12 h the respective AP protein mRNA levels were similarly affected in both turpentine- and DMSO-treated animals. The relative concentrations of MG, AGP, CPI, Hp and Fb mRNAs were, respectively, about 800%, 760%, 510%, 300% and 400% higher in the turpentine, and 500%, 610%, 720%, 250% and 450% higher in the DMSO-treated animals compared to the controls. In both turpentineand DMSO-treated rats the relative concentration of Al mRNA decreased to 85% of the control level. As can be seen after crossed immunoelectrophoresis on Fig. 3, the increase in AP protein mRNAs that was

Fig. 2. The effects of DMSO and turpentine administration on AP protein and albumin mRNA concenrations. The relative changes in concentration of the individual protein mRNAs were established by dot-blot analysis of RNAs isolated from turpentine-, DMSO-treated and control rats killed 12 h after treatment. Plasmids bearing inserts complementary to the indicated protein mRNAs were used: Al, albumin; AGP, ␣1 -acid glycoprotein; CPI, cysteine-proteinase inhibitor; Fb, ␥-fibrinogen; Hp, haptoglobin; MG, ␣2 -macroglobulin. The results are means from three separate experiments.

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Fig. 3. Crossed immunoelectrophoretic identification of the major AP proteins in the sera of turpentine- and DMSO-treated rats. Crossed immunoelectrophoresis was performed with sera isolated from control (A) and rats killed 24 h after the administration of DMSO (B) and turpentine (C) using polyspecific antibodies obtained from rabbits injected with sera of turpentine-treated rats. Proteins were stained with Coomassie Blue.

observed at 12 h was accompanied by an increase of AP proteins in the circulation by the 24th hour. Quantification of the electrophoregrams (Fig. 4) of sera isolated 24 h after DMSO and turpentine treatment when the AP proteins attained the maximal/plateau concentrations in the circulation revealed that the relative concentrations of MG, AGP, CPI and Hp were, respectively, about 820%, 780%, 540% and 310% higher after turpentine administration, and 510%, 630%, 710% and 280% higher after DMSO-treatment. The con-

centrations of Fb and Al were measured as described in Section 2. The relative concentrations of Fb were 380% and 420% greater in the sera of turpentine- and DMSO-treated rats, respectively (Fig. 4). The negative AP reactant albumin decreased to 90% and 93% in turpentine- and DMSO-treated rats, respectively (inset in Fig. 4). These results revealed that the degree of change of the serum levels of all of the examined proteins was similar to that of their corresponding mRNA concentrations. This finding is in agreement

Fig. 4. Summary of turpentine and DMSO-induced changes of relative protein concentrations in the serum. MG, AGP, CPI and Hp were identified by crossed immunoelectrophoresis, the areas under each immune-precipitated peak were measured and the turpentine- and DMSO-induced alterations expressed as the percent change relative to the respective control values (100%). The concentrations of Fb and Al were determined as described in the Methods section. The values are mean ± S.E. from three to five separate experiments. The inset shows the concentrations (mg/ml) of albumin determined in the sera of control, turpentine- and DMSO-treated rats. The results are means from three separate experiments.

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with the earlier observation that all of the available AP protein RNA templates are used in protein synthesis in the course of the AP response (Ševaljevi´c et al., 1989b, 1990, 1992; Glibeti´c et al., 1992). The AP response is a non-specific reaction of an organism to stress. Whereas different inducers of the AP response elicit a similar overall temporal course of AP protein production, different inducers promote reproducible and slightly different relative changes of individual AP protein concentrations in the circulation (Ševaljevi´c et al., 1989b, 1990, 1992; reviewed by Poznanovi´c et al., 1997). Comparison of the relative increases of AP reactant concentrations after turpentine- and DMSO-treatments revealed a degree of inducer-specific variations of AP protein patterns. For instance, whereas after turpentine-treatment MG and AGP were the main AP reactants, in response to DMSO treatment CPI was the major AP reactant. Slight variations of the AP protein pattern point to the existence of mechanisms that fine-tune the production of individual AP proteins best suited for neutralizing particular types of tissue and metabolic damage. The data obtained with DMSO are in agreement with the view that the non-specific homeostatic response to injury possesses a degree of specificity as the result of different targeting by diverse stressors and the subsequent activation of slightly different sets of mediators of inflammation. The results presented here show that DMSO is capable of eliciting the AP reaction. Awareness of this property should be kept in mind in view of its multifarious clinical, laboratory and industrial uses.

Acknowledgements This work was supported by the Research Science Fund of the Serbian Ministry of Science Technology and Development, Contract No. 1722.

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