Effects of Dimethylsulfoxide on Metabolism and Toxicity of Acetaminophen in Mice

Transcription

1 1618 Biol. Pharm. Bull. 29(8) (2006) Vol. 29, No. 8 Effects of Dimethylsulfoxide on Metabolism and Toxicity of Acetaminophen in Mice Mi Young YOON, a Sun Ju KIM, a Byung-Hoon LEE, a Jin-Ho CHUNG, a and Young Chul KIM*,a,b a College of Pharmacy, Seoul National University; and b Research Institute of Pharmaceutical Sciences, Seoul National University; San 56 1 Shinrim-Dong, Kwanak-Ku, Seoul , Republic of Korea. Received February 9, 2006; accepted May 16, 2006 Effects of dimethylsulfoxide (DMSO) on metabolism and toxicity of acetaminophen (APAP) were examined using male mice. A dose of DMSO (1 ml/kg, i.p.) inhibited the induction of APAP hepatotoxicity almost completely as indicated by changes in serum hepatotoxic parameters. Quantification of major APAP metabolites in plasma showed that APAP-glutathione (GSH), a conjugate generated via metabolic activation of APAP, was reduced significantly while APAP-sulfate and APAP-glucuronide, detoxified metabolites both produced directly from the parent drug, were increased in mice pretreated with DMSO. However, microsomal CYP2E1 activity measured with p-nitrophenol and p-nitroanisole as substrates was increased by DMSO treatment. Generation of APAP-GSH in microsomes from control mice was inhibited by DMSO in a dose-dependent manner. Lineweaver- Burk plot analysis indicated that the inhibition pattern produced by DMSO was competitive in nature. A g supernatant was reconstituted with the cytosolic fraction and microsomes from DMSO- or saline-treated animals. APAP-GSH production was increased significantly when the cytosolic fraction from saline-treated mice and/or microsomes from DMSO-treated mice were used. The results indicate that DMSO induces the enzyme activity responsible for oxidative metabolism of APAP, but its direct inhibitory effect on the enzymatic interaction with this drug decreases the overall production of a reactive metabolite, resulting in reduction of the hepatotoxicity. It is suggested that DMSO effects on metabolism of a xenobiotic would vary depending on its potential to inhibit the interaction of enzyme(s) and the xenobiotic. Key words acetaminophen; dimethylsulfoxide; glutathione; CYP2E1 Dimethylsulfoxide (DMSO) is frequently used as a solvent in various in vivo and in vitro experiments. The use of this solvent is favored especially when physicochemical properties of a test compound have not been well characterized such as in the case of natural products. But DMSO is not biologically inert. DMSO has numerous pharmacological effects, the most well-known of which is hydroxyl radical scavenging capacity. Therefore, it is suspected that toxicological implications of the interaction of DMSO with a xenobiotic, either directly or indirectly via body system, would be complex. Acetaminophen (APAP), a widely used analgesic-antipyretic, is detoxified rapidly through formation of sulfate and glucuronide conjugates. 1) However, at large doses this drug is increasingly converted into a reactive metabolite, N- acetyl-p-benzoquinonimine (NAPQI). The metabolic activation of APAP in human is mediated by CYP2E1, 1A2 and 3A4. 2,3) The relative contribution of each CYP isozyme to formation of the toxic metabolite is not clear, however, it has been generally accepted that CYP2E1 has the principal role in metabolic activation of this drug both in human and rodents. The reactive metabolite is normally detoxified by conjugation with glutathione (GSH), subsequently resulting in generation of APAP-mercapturate via APAP-cysteine. When generation of the reactive metabolite exceeds the availability of GSH for conjugation reaction, covalent binding of the metabolite to macromolecules may result, an event that correlates with induction of hepatic necrosis. 4) Therefore, the most important determinants for induction of APAP toxicity are the metabolic activities catalyzing its activation and the effectiveness of GSH conjugation reaction. It has been shown that induction of APAP hepatotoxicity may be decreased by a dose of DMSO in mice 5) and also in hamsters. 6) In mice DMSO was found to decrease covalent binding of APAP metabolites to hepatic protein, but the microsomal aniline hydroxylase activity was rather increased. 7) Accordingly, the authors suggested that the protective effect of DMSO was due to interaction of DMSO with free radicals or reactive APAP metabolites. Later studies revealed that the protection provided by DMSO was tissue-specific, which would exclude the possibility of involvement of a nonspecific mechanism such as scavenging of free radicals. 8) Also DMSO was shown to inhibit the biliary concentration of APAP-GSH and the microsomal dimethylnitrosamine N- demethylase activity. 9) Therefore, it appears that the protective effect of DMSO against APAP liver toxicity is associated with inhibition of CYP-dependent activation of this drug. Meanwhile, conflicting results were demonstrated in studies examining the effects of DMSO on metabolism and toxicity of other xenobiotics that are also metabolically activated by CYP2E1. For instance, DMSO was shown to enhance the hepatotoxicity of carbon tetrachloride (CCl 4 ), 10) while other studies demonstrated reduction of CCl 4 -induced hepatotoxicity in rats and mice. 11,12) Different studies showed that the hepatotoxicity of CCl 4 or its congener, chloroform (CHCl 3 ), was not altered by DMSO. 5,13) More recently Lind and colleagues 14,15) demonstrated that CHCl 3 hepatotoxicity was decreased by DMSO given as late as 24 h after CHCl 3, and suggested that the protective effect of DMSO could not be explained by a decrease in metabolic conversion of this substance to a reactive metabolite. The inconsistency in the effects of DMSO on metabolism and toxicity of xenobiotics suggests that this solvent may induce multiple actions on the metabolic reaction mediated by CYP2E1. In this study we examined the changes in generation of metabolic products from APAP and the resulting he- To whom correspondence should be addressed. youckim@snu.ac.kr 2006 Pharmaceutical Society of Japan

2 August patotoxicity in mice treated with DMSO, and correlated the results with its effects on the metabolic activation of this drug in vitro. The usefulness of DMSO as a solvent in various experiments could be enhanced by a better understanding of its effects on the hepatic metabolism of xenobiotics. MATERIALS AND METHODS Animals and Treatments Male ICR mice (25 35 g) were obtained from Dae Han Laboratory Animal (Seoul, Korea). The use of animals was in compliance with the guidelines established by the Animal Care Committee of this institute. All animals were housed in environmentally controlled rooms (22 2 C; 55 5% humidity) with a 12 h light/dark cycle for at least one week before use. Rodent chow and tap water were allowed ad libitum. Mice were fasted overnight in stainless steel wire bottomed cages before treatment with DMSO (0.4, 1.0, or 2.5 ml/kg, i.p.) diluted with normal saline. Control mice were treated with an equal volume of saline. APAP (250 mg/kg, i.p.) dissolved in distilled water was administered to mice 15 min following DMSO treatment. Volume of each injection was adjusted to 10 ml/kg. Chemicals APAP, NADH, b-nadph, 5,5 -dithio-bis(2- nitrobenzoic acid) (DTNB), GSH, 4-nitrocatechol, and aminopyrine were purchased from Sigma Chemical (St. Louis, MO, U.S.A.). p-nitrophenol was obtained from Aldrich-Chemie (Steinheim, Germany). Standard metabolites of APAP, such as APAP-glucuronide, APAP-sulfate, APAPmercapturate, APAP-cysteine and APAP-GSH, were kindly donated by McNeil Consumer Products (Fort Washington, PA, U.S.A.). All other chemicals and solvents used were reagent grade or better. Measurement of Serum Parameters Twenty four hours following APAP administration blood was collected by cardiac puncture on mice under light ether anesthesia. Blood samples were allowed to clot at room temperature followed by centrifugation. Activities of aspartate aminotransferase (AST) and alanine aminotransferase (ALT) in serum were determined employing the method of Reitman and Frankel. 16) Sorbitol dehydrogenase (SDH) activity was measured spectrophotometrically. 17) Blood urea nitrogen (BUN) was measured using a commercially available kit (Youngdong Pharmaceutical, Seoul, Korea). Determination of Plasma Concentrations of APAP Metabolites Plasma concentrations of APAP and its metabolites were measured using a HPLC method described elsewhere. 18) Briefly, plasma was mixed with an aliquot of acetonitrile containing theophylline as an internal standard. After extraction and centrifugation, the resulting supernatant was evaporated to dryness under nitrogen. The residue was diluted with distilled water as necessary before being injected into HPLC. APAP and its metabolites, APAP-GSH, APAP-cysteine, APAP-mercapturate, APAP-glucuronide and APAP-sulfate were separated in a reverse phase C 18 mbondapack column (30 cm 3.9 mm; Waters Associates, Milford, MA, U.S.A.). APAP and the metabolites, eluted with 1.8% aqueous acetic acid methanol H 2 O (66 : 9 : 100) at a flow rate of 1.5 ml/min, were monitored at 254 nm. Measurement of Hepatic GSH Levels Hepatic total GSH content was determined using an enzymatic recycling method. 19) Whole liver was homogenized in ice-cold 1 M perchloric acid with 2 mm EDTA followed by centrifugation. An aliquot of the supernatant was mixed with 0.3 mm NADPH and 6 mm DTNB. After addition of GSH reductase the absorbance was monitored at 412 nm. Measurement of DMSO in Plasma and Liver DMSO concentration in liver and plasma was determined using the method of Layman and Jacob. 20) Liver was homogenated in 1 M HClO 4 with 2 mm EDTA. After centrifugation, an aliquot of dimethylformamide was added as an internal standard. The supernatant was neutralized with 2 N KOH/3-[N-morpholino]propanesulfonic acid buffer before injection into GC equipped with a flame ionization detector. A 30 m DB5 capillary column (J&W Scientific, CA, U.S.A.) was used. Nitrogen was the carrier gas (30 ml/min), and air (300 ml/min) and hydrogen (30 ml/min) were utilized in the flame ionization detector. The detector temperature was set at 250 C, the injector at 240 C, and the column at 80 C. Assessment of Microsomal Enzyme Activities Mice were treated with DMSO (2.5 ml/kg, i.p.) before sacrifice at predetermined time points. Whole liver was homogenized in M KCl/50 mm Tris HCl, ph 7.4, with 1 mm EDTA. Homogenate was centrifuged at g for 20 min, and the supernatant was further centrifuged at g for 60 min. The microsomal pellet was suspended in the homogenizing buffer followed by recentrifugation at g for 60 min. The total CYP content was estimated from the CO difference spectrum. 21) Protein content was measured using the method of Lowry et al. 22) p-nitrophenol hydroxylase activity was determined by measuring the formation of p-nitrocatechol. 23) Aminopyrine N-demethylase and erythromycin N- demethylase activities were determined by quantifying the formaldehyde production. 24) p-nitroanisole O-demethylase was determined using the method of Shigematsu et al. 25) Determination of APAP-GSH Generation in Microsomes Generation of APAP-GSH was characterized using microsomes of control mice. A 0.5 ml reaction mixture containing 0.5 mg microsomal protein, NADPH (2.5 mm), GSH (5 mm), and varying concentrations of APAP and DMSO in 0.1 M phosphate buffer (ph 7.4) was incubated for 10 min at 37 C. Reaction was terminated by 0.1 ml ice-cold 2 M HCl. After addition of theophylline the mixture was centrifuged at rpm for 20 min. The supernatant was analyzed for APAP-GSH using the HPLC method described above. Generation of APAP-GSH in a Reconstituted g Supernatant Mice were treated with saline or DMSO (2.5 ml/kg, i.p.) 2h prior to sacrifice. The cytosolic fraction obtained after centrifugation of the g supernatant at g for 60 min was separated by decantation. The microsomal fraction was suspended in the homogenizing buffer followed by recentrifugation at g for 60 min. The microsomal pellet was resuspended and the protein content was determined. The microsomal suspension was mixed with the cytosolic fraction obtained after the first ultracentrifugation. The mixing ratio was determined by normalizing the microsomal protein content. APAP-GSH generation and its quantification were conducted employing the methods described above. APAP was added in the reaction mixture at a concentration of 0.5 mm.

3 1620 Vol. 29, No. 8 RESULTS Effect of DMSO on APAP-Induced Toxicity in Mice A single dose of APAP (250 mg/kg, i.p.) elevated ALT, AST, and SDH activities in serum markedly when measured 24 h after the treatment (Table 1). APAP at the dose used did not alter BUN. DMSO alone did not affect any parameters determined. The elevation of serum hepatotoxic parameters in APAP-treated mice was decreased effectively by a 15 min prior dose of DMSO. The induction of APAP hepatotoxicity was blocked almost completely by DMSO administered at a dose of 1 ml/kg. Different groups of mice were treated with DMSO 6 or 10 h after APAP, but the hepatotoxicity of APAP was not affected as determined by the changes in serum parameters (data not shown). Metabolism of APAP in Mice Pretreated with DMSO Changes in the plasma concentration of APAP and its metabolites are shown in Fig. 1. Concentrations of APAPglucuronide and APAP-sulfate, major detoxification products generated directly from APAP, were elevated in mice pretreated with DMSO. On the other hand, APAP-GSH, a conjugate of GSH and the reactive metabolite of APAP, was decreased significantly. The overall elimination of APAP was not changed by DMSO. Table 2 summarizes the area under the plasma concentration time curve (AUC ) of each metabolite. The AUC s of APAP-glucuronide and APAP-sulfate were Table 1. DMSO (ml/kg) Effect of DMSO Treatment on Toxicity Induced by APAP APAP (mg/kg) ALT AST SDH BUN (units/ml) (mg/dl) * * ** ** 36 9** ** ** 34 2** 21 2 Mice were treated with DMSO intraperitoneally 15 min prior to APAP. Each value represents the mean S.E. for five or six mice., Significantly different from mice treated with saline before APAP (Student s t-test, p 0.05, 0.01, respectively). Fig. 1. Effect of DMSO Treatment on Plasma Concentrations of APAP and the Metabolites Mice were treated with DMSO (2.5 ml/kg, i.p.) or saline 15 min prior to APAP (250 mg/kg, i.p.). Each value represents the mean S.E. for four mice. Filled triangle indicates mice pretreated with DMSO; filled circle, pretreated with saline., Significantly different from mice treated with saline before APAP (Student s t-test, p 0.05, 0.01, respectively).

4 August increased approximately by 50%, but the AUCs of APAP- GSH and APAP-cysteine were reduced markedly in mice treated with DMSO. Effect of DMSO on Hepatic Microsomal Drug Metabolizing Enzyme Activities Hepatic metabolizing enzyme activities were measured in microsomes from mice treated with DMSO (2.5 ml/kg, i.p.) (Table 3). p-nitrophenol hydroxylase and p-nitroanisole O-demethylase activities, in which CYP2E1 plays a major role, were increased as early as at 2 h following the treatment. But aminopyrine N-demethylase and erythromycin N-demethylase activities were decreased at 4 to 6 h after DMSO treatment. It was noted that the induction of CYP2E1 activity was significant while the Effect of DMSO Treatment on AUCs of APAP and the Metabo- Table 2. lites Metabolites Control (mg h/ml) DMSO (% control) APAP (115) APAP-glucuronide * (144) APAP-sulfate * (157) APAP-GSH *** (47) APAP-cysteine * (69) APAP-mercapturate (100) Mice were treated intraperitoneally with DMSO (2.5 ml/kg) 15 min prior to APAP (250 mg/kg, i.p.). Each value represents the mean S.E. for four mice. AUC was calculated from plasma concentrations vs. time curve using a trapezoidal method., Significantly different from control mice treated with saline before APAP (Student s t-test, p 0.05, 0.001, respectively). generation of APAP-GSH was inhibited in mice treated with DMSO (Fig. 1). The CYP activities returned to normal levels in 24 h except a small increase in p-nitrophenol hydroxylase. GSH and DMSO Levels in Blood and Liver Changes in hepatic GSH levels in mice treated with APAP (250 mg/kg, i.p.) and/or DMSO (2.5 ml/kg, i.p.) are shown in Fig. 2. DMSO alone did not alter the hepatic GSH levels, but the GSH depletion induced by a toxic dose of APAP was significantly inhibited in mice pretreated with DMSO. The concentration of DMSO in blood and liver reached peak levels at 1 to 2 h after the treatment, and declined linearly with a halflife of approximately 2 h (Fig. 3). Inhibition of Microsomal APAP-GSH Generation by DMSO The effect of DMSO on the production of APAP- GSH was determined in microsomes of control mice. Generation of APAP-GSH was inhibited by addition of DMSO to the reaction mixture in a dose-dependent manner. The inhibition of APAP-GSH generation was characterized kinetically. The double reciprocal plot of GSH conjugate formation vs. APAP concentration revealed that the inhibition pattern produced by DMSO was competitive in nature (Fig. 4). The V max of the enzyme responsible for generation of APAP-GSH was 1.39 nmol/min/mg protein; K m, 0.44 mm. The plot of the apparent K m obtained against the DMSO concentration used gives a K i of 11.8 mm. APAP-GSH Generation in a Reconstituted g Supernatant A reconstituted g supernatant was used to estimate the effect of DMSO injected into animals on the hepatic generation of APAP-GSH. Generation of APAP-GSH Table 3. Effect of DMSO Pretreatment on Microsomal Enzyme Activities p-nitrophenol p-nitroanisole Aminopyrine Erythromycin Time CYP hydroxylase O-demethylase N-demethylase N-demethylase (h) (nmol/mg protein) (nmol product formed/min/mg protein) *** * ** ** ** *** * ** ** * Mice were treated with DMSO (2.5 ml/kg, i.p.) and sacrificed at the predetermined time points. Each value represents the mean S.E. for four pooled samples, each made of livers from four mice. Therefore, a total of sixteen mice were used per group.,, Significantly different from control mice (t 0 h) (one-way ANOVA followed by Dunnett s t-test, p 0.05, 0.01, 0.001, respectively). Fig. 2. Effect of DMSO and/or APAP on Hepatic GSH Levels Mice were treated with DMSO (2.5 ml/kg, i.p.) and/or APAP (250 mg/kg, i.p.). Each value represents the mean S.E. for four mice. Open circle indicates mice treated with DMSO only; filled circle, treated with saline before APAP; filled triangle, treated with DMSO before APAP.,, Significantly different from mice treated with saline and APAP (Student s t-test, p 0.05, 0.01, respectively). Fig. 3. Disappearance of DMSO from Plasma and Liver Mice were treated with DMSO (2.5 ml/kg). Each value represents the mean S.E. for four mice. Filled circle represents plasma concentration; filled triangle, hepatic concentration.

5 1622 Vol. 29, No. 8 Fig. 4. Lineweaver Burk Plot of APAP-GSH Formation vs. APAP Concentration in Microsomes with Different Concentrations of DMSO Each point represents the mean of five or six pooled samples, each made of livers from four mice. Open circle indicates 0 mm DMSO; filled circle, 3 mm; open square, 5 mm; filled square, 10 mm. The inlet at the left shows the plot of the apparent K m obtained against the DMSO concentration used; it gives a K i of 11.8 mm. Fig. 5. APAP-GSH Generation in a Reconstituted g Supernatant Mice were treated with saline or DMSO (2.5 ml/kg, i.p.) 2 h prior to sacrifice. [DM] and [DC] indicate microsomes and cytosol from DMSO-treated mice; [CM] and [CC], microsomes and cytosol from saline-treated mice, respectively. Each value represents the mean S.E. for four pooled samples, each made of livers from four mice. Values with different letters (a, b, c) are significantly different one from another (one-way ANOVA followed by Newman Keuls multiple range test, p 0.05). was significantly greater in the preparation containing microsomes from DMSO-treated mice [DM] (Fig. 5), which was consistent with the induction of microsomal p-nitrophenol hydroxylase and p-nitroanisole O-demethylase activities. But the cytosolic fraction obtained from DMSO-treated mice [DC] decreased the APAP-GSH production when mixed with microsomes from either saline-treated mice [CM] or DMSOtreated mice [DM]. The results indicated that the presence of DMSO in the cytosolic fraction was directly responsible for the depression of APAP-GSH generation despite the induction of CYP2E1 activity. DISCUSSION The frequent use of DMSO as a solvent in laboratories and industries indicates that a better understanding of the effects of this substance on metabolism of xenobiotics is necessary. In 1982 Morgan et al. demonstrated that ethanol oxidation in microsomes of rabbits treated chronically with ethanol was inhibited by DMSO. 26) Later studies showed that DMSO decreased the microsomal dimethylnitrosamine demethylase activity in rats 27) and also in mice. 9) DMSO was shown to inhibit the activities of several CYP isozymes such as 2E1, 2C9, 2C19 and/or 3A4 in human liver. 28,29) In contrast, Zangar et al. 30) reported that DMSO increased CYP2E1 and 3A proteins, which appeared to be in accordance with the earlier observation that aniline hydroxylase activity was increased in mice pretreated with DMSO. 7) Recently induction of mrna levels of CYP isoforms including 2E1, 3A4 and 1B1 by DMSO has been demonstrated. 31) Therefore, the results from studies examining the DMSO effects on the hepatic CYP-dependent metabolizing activities are not consistent. In this study we determined the effects of DMSO on metabolic conversion of APAP in vivo as well as in vitro, and the significance of their interaction in induction of the hepatotoxicity. Treatment of mice with DMSO 15 min prior to APAP resulted in an almost complete inhibition of the hepatotoxicity. But DMSO administered 6 or 10 h after APAP did not affect the toxicity of this drug. APAP and its metabolites disappeared from blood almost completely in 6 h. This result suggests that the protective action provided by DMSO would be most probably associated with its effects on metabolic activation or detoxification of this drug. But the hepatic microsomal enzyme activities mediating the metabolic conversion of APAP into a toxic intermediate were elevated significantly by DMSO during the period the metabolism of APAP actively took place. p-nitrophenol hydroxylase and p-nitroanisole demethylase activities, in which CYP2E1 plays a major role, 23,32) were increased from 2 h following the treatment. This is in accordance with earlier studies showing an increase in the hepatic aniline hydroxylase activity, 7) mrna levels, 31) and expression of CYP2E1 30) by DMSO administration to rodents. In contrast, DMSO pretreatment decreased aminopyrine N-demethylase and erythromycin N-demethylase activities, in which CYP1A2 and/or 3A are involved. These results suggest that DMSO induces variable effects on the activities of different CYP isozymes. In mice treated with DMSO the plasma concentration of GSH conjugates was reduced significantly. The reduction of APAP-GSH concentration did not agree with the enhancement of CYP2E1 activity in microsomes of DMSO-pretreated mice, but coincided well with the inhibition of APAP hepatotoxicity. Hepatic GSH concentration was not altered by DMSO alone, and also the GSH depletion by APAP challenge was decreased in mice pretreated with DMSO, indicating that the reduction of APAP-GSH concentration could not be accounted for by a change in the GSH availability. In contrast, APAP-glucuronide and APAP-sulfate conjugates, the detoxicification products generated directly from APAP, were

6 August increased, showing that metabolism of this drug via these pathways was enhanced by DMSO. The effect of DMSO on glucuronide and sulfate conjugation reactions was not determined in this study, but it appears that the inhibition of oxidative metabolism by DMSO results in distribution of a greater portion of this drug to the detoxification pathways via generation of sulfate and glucuronide conjugates. CYP1A2 and 3A4 are also implicated in the metabolic activation of APAP in human and rodents. 2,3) It has been generally accepted that the role of CYP1A2 and 3A4 in generation of NAPQI is very small compared with that of CYP2E1. 3,33,34) In this study a dose of DMSO to mice decreased the microsomal CYP1A2 and 3A activities determined with aminopyrine and erythromycin as substrates. The contribution of inhibition of CYP1A2 and/or 3A activities by DMSO to the reduction of generation of a reactive metabolite from APAP in vivo is not known. But the apparent decrease in APAP-GSH concentrations in blood despite the induction of CYP2E1, the major isozyme responsible for generation of a reactive metabolite, suggests that a change in the microsomal activities of CYP isozymes including CYP1A2 and 3A does not play a crucial role in the reduction of metabolic activation of APAP in vivo. The effect of DMSO on generation of APAP-GSH was characterized kinetically using microsomes from control mice. Formation of APAP-GSH is a virtually two step reaction; generation of NAPQI from APAP and conjugation of NAPQI with GSH. It has been shown that both nonenzymatic reaction and glutathione S-transferases (GSTs)-mediated reaction are involved in the conjugation reaction, and that at high concentrations of GSH and NAPQI, rapid nonenzymatic detoxification predominates. 35) In that study the rate of conjugate formation of GSH with NAPQI was demonstrated to by far exceed the rate of APAP-GSH generation from APAP, 36) indicating that oxidation of APAP to the reactive metabolite is the rate-limiting step in generation of APAP-GSH. Therefore, the formation of APAP-GSH may reflect the production of the reactive metabolite from this drug when GSH concentrations are maintained. 37) In this study the microsomal APAP-GSH production was inhibited competitively by addition of DMSO. This result is in agreement with an observation that DMSO inhibited competitively the microsomal N- demethylation of dimethylnitrosamine, 9) which is also a CYP2E1 substrate. The K i was determined to be 11.8 mm. The concentration of DMSO in liver reached a peak of approximately 30 mm at 2 h after administration at a dose of 2.5 ml/kg, i.p., which suggests a significant inhibition of the enzymatic reaction in whole animals. This appears to explain the decrease in the plasma concentration of thioether conjugates of APAP despite the induction of CYP2E1 activity in animals pretreated with DMSO. It is suggested that DMSO decreases metabolic activation of APAP via its direct inhibitory effects on the interaction of APAP with the enzyme site in animals. In order to estimate the effect of DMSO administered to mice on the metabolic activation of APAP in liver, generation of APAP-GSH was determined using a reconstituted g supernatant. Mice were sacrificed 2 h following saline or DMSO treatment when hepatic DMSO levels reached peak concentrations. The cytosolic fraction and microsomes were separated followed by remixing to reconstitute a g supernatant. APAP-GSH generation was greater in a g supernatant containing microsomes from DMSO-treated mice than in a g supernatant containing control microsomes, which was in agreement with the induction of microsomal CYP2E1 activity. But when the cytosolic fraction from DMSO-treated mice was used in a reconstituted g, the APAP-GSH production was decreased markedly. The results indicated that the presence of DMSO in the site where the enzyme reaction took place was directly responsible for the depression of metabolic conversion of APAP into the reactive metabolite. It is suggested that the inhibitory effect of DMSO on the CYP-mediated reaction overrules the increase in CYP2E1 activity, leading to an overall reduction of the metabolic activation and the resulting hepatotoxicity of APAP. The present results indicate that DMSO induces complex effects on metabolism of xenobiotics. While this substance has variable effects on the activities of different CYP isozymes, its presence in the enzyme sites produces direct inhibitory effects on the metabolic reaction mediated by the enzymes. The relative affinity of DMSO and a substrate for the enzymes would play a crucial role in the consequence of their interactions. Due to the hydrophobic nature of the CYP enzyme site, 38) the competition between DMSO and a substrate for the enzyme site would be essentially influenced by the hydrophobicity of each compound. The inconsistency in the effects of DMSO on the toxicity of various xenobiotics observed in earlier studies could be accounted for, at least partly, by a difference in the hydrophobicity of the substances. We have recently observed that DMSO does not affect the metabolic degradation of CCl 4 while that of CH 2 Cl 2, which is far less lipophilic than CCl 4, is inhibited significantly (unpublished data). 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