A labile sulfur in trisulfide affects cytochrome P-450 dependent lipid peroxidation in rat liver microsomes

Toxicology Letters 99 (1998) 191 – 198

A labile sulfur in trisulfide affects cytochrome P-450 dependent lipid peroxidation in rat liver microsomes Yuki Ogasawara, Shinji Isoda, Shinzo Tanabe * Department of Hygienic Chemistry, Meiji College of Pharmacy, 1 -22 -1, Yato-cho, Tanashi-shi, Tokyo 188, Japan Received 25 May 1998; received in revised form 27 July 1998; accepted 28 July 1998

Abstract The effects of trisulfide derivatives were studied on cytochrome P-450-dependent lipid peroxidation using rat liver microsomal systems. Cytochrome P-450-dependent lipid peroxidation was induced by carbon tetrachloride or tert-butylhydroperoxide and was evident by an increase in thiobarbituric acid-reactive substances (TBA-RS) and oxygen consumption. In these cytochrome P-450-dependent lipid peroxidation systems, pretreatment of microsome with trisulfide derivatives (cystine trisulfide and thiocyclam) significantly inhibited TBA-RS formation and oxygen consumption compared with disulfide or thiol analogs (cystine, nereistoxin, or cysteine). The labile sulfur contained in trisulfide disappeared during incubation with liver microsomes. In the CCl4-induced lipid peroxidation system, the cytochrome P-450 level and NAD(P)H-cytochrome P-450 reductase activity were significantly decreased by the addition of trisulfide derivatives. Therefore, in cytochrome P-450-dependent lipid peroxidation system, the potential effects of trisulfide appear to be mediated via enzyme inhibition. These results suggest that pretreatment of the trisulfide derivatives may affect the toxic function of exogenous xenobiotics or drugs, which are reduced by the liver enzyme cytochrome P-450 to radical species. © 1998 Elsevier Science Ireland Ltd. All rights reserved. Keywords: Trisulfide; Lipid peroxidation; Cytochrome P-450; Rat liver microsomes; Cystine trisulfide; Thiocyclam

1. Introduction

Abbre6iations: TC, thiocyclam; CT, cystine trisulfide; tBuOOH, tert-butylhydroperoxide; Asc, ascorbate; TBA-RS, thiobarbituric acid-reactive substances; NT, nereistoxin. * Corresponding author. Tel.: +81 424 958529; fax: +81 424 958529.

Recently, biological characterization or potentialities of many trisulfide derivatives have been studied (Golik et al., 1987; Zein et al., 1988; Litaudon and Guyot, 1991; Jespersen et al., 1994). Some natural products containing trisulfide structure act as potent antitumor or antifungal agents. Furthermore, it was indicated that glutathione

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trisulfide was recognized and reduced by glutathione reductase (Moutiez et al., 1994). Trisulfide derivatives have a labile sulfur atom covalently bound to other sulfur atoms of a thiol compound. It is estimated that this additional sulfur atom has a significant effect on the potentiality and metabolism of trisulfide derivatives. In previous studies, we have found that the same sulfur atoms commonly exist in mammalian sera (Togawa et al., 1992; Ogasawara et al., 1993) and tissues (Ogasawara et al., 1994). This labile sulfur species is referred to as ‘bound sulfur’, which is defined as divalent sulfur that is easily liberated as sulfide by reduction with excess thiols. In many bound sulfur-containing compounds, the sulfur is labile, readily coming out of the structure to be reduced to sulfide by reducing agents. The following bound sulfur-containing compounds have been shown to be involved in biological systems: elemental sulfur (S8), hydrodisulfides frequently called persulfides (RSSH), polysulfides (R – Sn – R) where n must be 3 or greater (Wood, 1982; Toohey, 1989). It was also suggested that bound sulfur-containing compounds in biological systems are in a equilibrium with the charged and uncharged state (Ogasawara et al., 1993). In this study, we used trisulfide derivatives as models of bound sulfur-containing compounds, which were comparably stable. Although the physiological function of these sulfur species is not well understood, biological sulfur that has reduced oxidation states is thought to have some important function in vivo (Toohey, 1989). On the other hand, thiols are recognized for their radical-scavenging role in protection against cellular oxidative stress (Winterbourn, 1993; Buttke, 1994) and many biological thiols, such as glutathione (GSH), cysteine (Thuchida et al., 1985; Motoyama et al., 1989) N-acetylcysteine, and metallothioneine (Shitaishi et al., 1982) are known as antioxidants. Everett et al. (1992) indicated that the structurally related disulfur analogs or perthiols (persulfide, RSSH) have significantly different free radical-scavenging and acid/base properties compared to thiols because of relative differences in S–H bond energies (Everett et al., 1992; Wardman et al., 1994). The outer sulfur

atom in a persulfide residue is a typical bound sulfur, and persulfides have been found in mammalian serum (Ogasawara et al., 1993). Thus, the action of bound sulfur in biological systems was investigated using trisulfide derivatives in an attempt to elucidate some of the roles of bound sulfur in vivo. The objectives of the present study were to determine the effects of trisulfide derivatives containing a bound sulfur on lipid peroxidation in liver microsomes and to evaluate the action of bound sulfur in this process.

2. Materials and methods

2.1. Chemicals 2-Thiobarbituric acid, HPLC-grade ethanol and carbon tetrachloride were supplied by Merck (Darmstadt, Germany). tert-Butyl hydroperoxide (t-BuOOH), cysteine, cystine, b-NADPH and butylated hydroxytoluene were purchased from Sigma (St. Louis, MO). Thiocyclam, nereistoxin, ferrous sulfate, and dithiothreitol were purchased from Wako (Osaka, Japan). Cystine trisulfide was prepared as described by Fletcher and Robson (1963). Standard solutions of the analytes and reagents for bound sulfur determination were prepared according to our previous report (Ogasawara et al., 1993). All other reagents were commercial products of the highest available purity.

2.2. Animals Male Wistar rats weighing between 250 and 280 g were used in all experiments. Rats were housed in a temperature-controlled (23°C) and light-controlled (12-h cycle) animal room. The animals had ad libitum access to water and standard laboratory chow CE-2 (Clea, Japan) prior to experiments. All animals received humane care as the guideline of our institution for animal experiments founded on the National Institutes of Health Guide for Care and Use of Laboratory Animals.

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2.3. Preparation of rat li6er microsomes Rats were anaesthetized intraperitoneally with pentobarbital in accordance with institutional guidelines. Livers were removed following thorough in situ perfusion through the portal vein with ice-cold 10 mM phosphate-buffered 0.14 M NaCl (PBS, pH 7.4). A 10% (w/v) liver homogenate in 154 mM KCl was prepared using a Potter-Elvehjem homogenizer with a teflon pestle. The homogenate was centrifuged once at 10000× g, at 4°C for 20 min and the supernatant was recentrifuged at 105000×g, at 4°C for 60 min. The microsomal pellet thus obtained was washed twice with 1.15% KCl, and final microsomal precipitates were suspended in 50 mM Tris – HCl buffer (pH 7.4). The protein content of the microsomal suspension was determined by the method of Lowry et al. (1951) and adjusted to 5 mg protein/ml of suspension. Microsomes were freshly prepared before each batch of experiments.

2.4. Measurement of TBA-RS and oxygen consumption TBA-reactive substances (TBA-RS) were estimated using the conventional method (Beuge and Aust, 1978) after the microsomal lipid peroxidation was stopped by the addition of trichloroacetic acid. The oxygen concentration of the incubation mixture was monitored using a Clark-type oxygen electrode (YSI 5331 model, Yellow Spring) in a closed glass vessel protected from light, maintained at 37°C, and equipped with a stirrer (Estabrook, 1967). Reactions were initiated via the sequential addition of an inducer (Fe2 + , tBuOOH and CCl4) to each of the microsomal suspensions and changes in oxygen tension were recorded.

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Trisulfide derivatives and reference test samples were preincubated at 37°C for 5 min in a microsomal suspension. Peroxidation induced by the Fe2 + -ascorbic acid (Asc) was carried out by incubating the microsomal suspension (1 mg protein/ ml) at 37°C for 10 min in 0.1 M phosphate buffer (pH 7.4) containing 0.5 mM Asc and 20 mM ferrous sulfate (Kagan et al., 1990). Peroxidation induced by t-BuOOH was carried out by incubating the microsomal suspension (0.8 mg protein/ ml) at 37°C for 30 min in 0.1 M phosphate buffer (pH 7.4) containing 1 mM t-BuOOH (Cadenas and Sies, 1982). Peroxidation induced by CCl4 was carried out by incubating the microsomal suspension (1 mg protein/ml) at 37°C for 30 min in 0.15 M phosphate buffer (pH 7.5) containing 0.25 mM NADPH, 9 mM CCl4 and 1 mM EDTA (Masuda and Murano, 1977). In all the systems, the amount of lipid peroxidation was determined via measurement of the concentration of TBA-RS, and the rate of oxidation was followed by monitoring the oxygen consumption.

2.6. Effect of trisulfide deri6ati6e on microsomal enzymes Microsomal suspension (1 mg protein/ml) were incubated at 37°C in the presence or absence (control) of 100 mM trisulfide derivatives and reference test samples. At 30 min after the addition of test sample (or buffer solution for control), portion of mixture was withdrawn to assay for cytochrome P-450 content and NAD(P)H cytochrome P-450 reductase activity. NAD(P)H-Cytochrome P-450 reductase activity was measured spectrophotometrically as described by Masters et al. (1967), and the content of cytochrome P-450 was determined using the method of Omura and Omura and Sato (1964).

2.7. Other methods 2.5. Lipid peroxidation systems The effects of trisulfide derivatives were tested using the three models of rat liver microsome systems indicated below, and compared with related thiol, disulfide analogs.

The consumption of bound sulfur in the lipid peroxidation systems was monitored using an HPLC method originally developed for the analysis of biological bound sulfur. Aliquots (500 ml) were taken from the microsomal incubation mix-

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tures and transferred to snap-cap tubes in an ice-cold bath. After the addition of 500 ml of 20 mM dithiothreitol (DTT) to the sample, the mixture was incubated at 37°C for 15 min. According to previously described methods, the reduced reaction mixture was subjected to flow gas dialysis followed by derivatization reaction and chromatographic separation with fluorometric detection (Ogasawara et al., 1993).

3. Results To elucidate the action of bound sulfur in biological systems, two different trisulfide compounds containing a bound sulfur were examined in lipid peroxidation systems, and the results compared with those for the reference thiol and disulfide analogs (Fig. 1). Lipid peroxidation was induced in rat liver microsomes in three different systems. Table 1 shows the extent of inhibition of TBA-RS formation by 100 mM trisulfide derivatives, reference compounds in the incubation mixture. In the Fe2 + -Asc, CCl4 and t-BuOOHinduced systems, lipid peroxidation was inhibited by approximately 19, 30 and 15% with cystine trisulfide (CT), and 3.5, 48 and 35% with thiocyclam (TC), respectively. The inhibitory effect of TBA-RS formation by cystine trisulfide and thiocyclam were significantly different from that observed using the same concentration of each reference disulfide or thiol in each system. The effects of thiocyclam and disulfide analog, nereis-

Fig. 1. Trisulfide derivatives and reference thiol and disulfide compounds. *Bound sulfur.

Fig. 2. Microsomal oxygen consumption induced by t-BuOOH in presence of trisulfide derivatives. Control, without test sample; blank, without initiator and test sample; TC, thiocyclam; NT, nereistoxin. Each test sample (100 mM) was preincubated with microsomes for 5 min prior to the addition of t-BuOOH(1 mM) as indicated by arrow (0 min). The reaction was carried out at 37°C. Vertical division indicates concentration of oxygen in the closed system relative to an air saturated solution.

toxin, on the rate of oxygen consumption in the peroxidation system induced by t-BuOOH are shown in Fig. 2. A microsomal suspension was initially saturated with air, and preincubated with the test sample. After the addition of t-BuOOH, the oxygen concentration in the mixture was immediately traced using an oxygen electrode. Although 100 mM of thiocyclam suppressed consumption of oxygen by 40% compared to the control, the same concentration of nereistoxin increased the yield of lipid peroxidation in the t-BuOOH-induced system (Fig. 2). Fig. 3 shows the effects of varying the concentrations of either cystine trisulfide (A) or thiocyclam (B) on TBA-RS formation induced by the t-BuOOH and CCl4-induced systems. Both cystine trisulfide and thiocyclam effectively suppressed oxidation in a concentration-dependent manner. To examine the antioxidant mechanism of the trisulfide derivatives, the consumption of bound

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Fig. 3. Effect of cystine trisulfide (A) and thiocyclam (B) on CCl4 or t-BuOOH-induced lipid peroxidation in rat liver microsomes. Incubation was performed at 37°C for 30 min, and peroxidation was then determined by TBA-RS test. CCl4 system, open circle; t-BuOOH system, closed circle. Results are mean of triplicate determinations taken from a representative experiment of two separate assays. Percentage is calculated after subtracting blank (no inducer) level of peroxidation: 100% peroxidation is equal to 6.0 nmol MDA/mg protein with CCl4, and 11.0 nmol MDA/mg protein with t-BuOOH.

sulfur in trisulfide during the various phases of lipid peroxidation was examined. The concentration of bound sulfur contained in spiked thiocyclam decreased during incubation in the CCl4 and t-BuOOH-induced lipid peroxidation systems. However, no significant differences were obtained between the addition and no addition of inducer (Fig. 4). These observations suggest that the influence of exogenous bound sulfur are not alone due to self-oxidation caused by radical trapping in the CCl4 and t-BuOOH-induced lipid peroxidation systems. Following this evaluation, the effects of trisulfide derivatives on the enzymatic systems related to free radical generation from CCl4 or t-BuOOH were investigated. As shown in Table 2, incubation with the trisulfide derivatives decreased both the cytochrome P-450 concentration and NAD(P)H-cytochrome P-450 reductase activity. In particular, approximately 50 – 70% of cytochrome P-450 rapidly disappeared with thiocyclam or cystine trisulfide. When the same concentration of nereistoxin, cysteine and

cystine was added to the microsomes, no significant decrease in either the cytochrome P-450 level or NAD(P)H-cytochrome P-450 reductase activity was observed.

4. Discussion In previous studies, we have shown that reduction-labile sulfur atoms are abundant in mammalian tissue and have defined these as ‘bound sulfur’ (Ogasawara et al., 1993, 1994). Bound sulfur appears to be derived from cysteine and a significant proportion that is produced from cystine by g-cystathionase is oxidized to sulfate (Stipanuk, 1986). However, the oxidation process of these reduction-labile sulfur atoms remain unclear. Although the physiological form of bound sulfur has yet to be identified, the biological characterization or activities of many bound sulfurcontaining trisulfide derivatives has recently been reported (Golik et al., 1987; Zein et al., 1988;

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Fig. 4. Depletion of bound sulfur in trisulfide during CCl4 and t-BuOOH-induced lipid peroxidation in rat liver microsomes. After the addition of thiocyclam (100 mM), rat liver microsomes were incubated at 37°C in the presence (closed triangle) or absence (open triangle) of CCl4, and in the presence (closed circle) or absence (open circle) of t-BuOOH. Residual concentration of bound sulfur was determined at indicated intervals.

Litaudon and Guyot, 1991; Jespersen et al., 1994; Moutiez et al., 1994). In the present study, in which two trisulfide derivatives were used as the low-molecular weight bound sulfur component, the potency of bound sulfur was evaluated in various lipid peroxidation systems. In CCl4 and t-BuOOH-induced systems, trisulfide containing a bound sulfur inhibited lipid peroxidation more effectively than the reference thiol or disulfide compounds (Table 1). The antioxidant effects of thiocyclam in the CCl4 and t-BuOOH system were significantly higher than that of cystine trisulfide. However, little effect on

lipid peroxidation induced by Fe2 + -Asc was observed with thiocyclam. It is evident that the significant inhibitory effects of trisulfides occur on cytochrome P-450-dependent lipid peroxidation. The Fe2 + -Asc system contains ferrous ion and a reducing agent, ascorbate. Such a combination is known to function as an inducer of oxidative stress by non-enzymatic stimulation of the production of oxygen radicals which react with the microsomal lipids. Therefore, as the inhibitory effect on lipid peroxidation induced by Fe2 + -Asc is caused by the direct interaction with free radical species, it is unlikely that trisulfide can act as a direct radical scavenger. The primary reaction of the CCl4 and tBuOOH systems is the one-electron enzymatic reduction of carbon tetrachloride (Slater and Saywyer, 1971) or t-BuOOH (Minnotti, 1989; Kagan et al., 1990) by the cytochrome P-450 present in liver microsomes. No significant difference in the fate of bound sulfur in trisulfide during lipid peroxidation was observed in the CCl4 and tBuOOH systems (Fig. 4). The rapid decrease in the concentration of the bound sulfur by 20–30% during microsomal incubation in the absence of peroxidation inducers suggests that parts of the bound sulfur in trisulfide are converted to another form before oxidative alteration by reacting with the microsomal components. Thus, it is estimated that the effects of trisulfide against lipid peroxidation depend on the inactivation for the microsomal enzymatic systems to generate radicals. To clarify the inhibitory effect of trisulfide, changes in the cytochrome P-450 level and cytochrome

Table 1 Inhibitory effects of trisulfide derivatives and referential compounds on lipid peroxidation induced by various systems Induced systems

Fe2+-Asc CCl4 t-BuOOH

Inhibition (%)a TC

NT

CT

Cystine

Cysteine

3.69 0.82 47.89 1.97 34.89 4.66

−1.691.31 2.09 1.00 3.690.67

18.9 9 5.56 29.8 9 5.90 14.4 91.72

8.6 9 6.15 6.2 92.89 4.7 9 5.15

1.9 94.72 10.0 93.76 5.1 9 2.37

TC, thiocyclam; NT, nereistoxin; CT, cystine trisulfude. Microsomal suspensions were preincubated at 37°C for 5 min in the presence or absence (control) of 100 mm test samples, and each assay of lipid peroxidation was performed at 37°C as described in Section 2. a (%) Inhibition of the accumulation rate of TBA-RS as compared to controls performed in parallel.

Y. Ogasawara et al. / Toxicology Letters 99 (1998) 191–198 Table 2 Effect of trisulfide derivatives and reference compounds on cytochrome P-450 and cytochrome P-450 reductase in rat liver microsomes Recovery

None Thiocyclam Nereistoxin Cystine trisulfide Cysteine Cystine

Cytochrome P450

Cytochrome P-450 Red.a

100 32.8 87.5 55.9 93.7 85.0

100 82.2 102.2 88.0 100.7 96.4

Assays were carried out as described in Section 2. Values are relative to control ( =100) and reported as means from two independent experiments. a NADPH cytochrome P-450 reductase.

P-450 NADPH reductase activity were examined in the incubation mixtures that contained trisulfide derivatives. Comparison with the reference, thiol or disulfide compounds, indicated that bound sulfur in trisulfide inactivates liver microsomal cytochrome P-450 (Table 2), which plays an essential role in the lipid peroxidation induced by CCl4 and t-BuOOH. It is known that diethyldithiocarbamate (DDC) inhibits the CCl4-induced lipid peroxidation of rat microsomes (Masuda and Murano, 1977; Freundt et al., 1981), and the NADPH-induced lipid peroxidation in mouse liver microsomes (Kulkarni and Hodgson, 1981). DDC is metabolized in vivo to carbon disulfide (Prickett and Johonston, 1953; Stro¨mme, 1965) and then further to active sulfur atoms (bound sulfur), which bind to the microsomal proteins, resulting in the suicidal loss of cytochrome P-450 (De Matteis, 1974; Dalvi et al., 1974). Although it is still unclear whether the inhibitory effect of DDC on the lipid peroxidation is due to its metabolites, it is possible that bound sulfur produced from DDC plays an essential role in the process. Our data indicate that bound sulfur in trisulfide have same potentiality as DDC and react with microsomal components on the lipid peroxidation induced by CCl4. Therefore, the interaction between bound sulfur and cytochrome

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P-450 should be investigated to clarify the active metabolites of compounds containing sulfur atoms, which can be converted to bound sulfur. Our results represent the first attempt at the elucidation of the inhibitory effect of bound sulfur in biological peroxidation systems. The above results clearly indicate that pretreatment of trisulfide containing a bound sulfur effectively inhibits cytochrome P-450-dependent lipid peroxidation in vitro, and this pretreatment may decrease the toxicity of exogenous xenobiotics or drugs, which are reduced by the cytochrome P450 to radical species. Further studies are necessary to elucidate whether the pretreatment of trisulfide derivatives inhibits cytochrome P-450dependent lipid peroxidation in vivo.

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