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Relative hepatotoxicity of substituted thiobenzamides and thiobenzamide-S-oxides in the rat
TOXICOLOGY
AND APPLIED
Relative
PHARMACOLOGY
(1980)
Hepatotoxicity of Substituted Thiobenzamides Thiobenzamide-S-oxides in the Rat1
ROBERT P. HANZLIK,~ Department
55, 260-272
of Medicinal
Chemistry,
Received
JOHN
R.
CASHMAN,
of Pharmacology Kansas 66045
and Department Lawrence.
November
12, 1979; accepted
AND
GEORGE and Toxicology,
and
J. TRAIGER University
of Kansas.
June 2, 1980
Relative Hepatotoxicity of Substituted Thiobenzamides and Thiobenzamide-S-oxides in the Rat. HANZLIK, R. P., CASHMAN, J. R., AND TRAIGER, G. J.(1980). Toxicol. Appl. Pharmacol. 55, 260-272. The effect of substituents on the relative hepatotoxicity of various derivatives of thiobenzamide (TB) and thiobenzamide-S-oxide (TBSO) has been investigated in the rat. For five para-substituted thiobenzamides (p-XCGH,CSNH,: X = CH,O, CH,, H, Cl, CF,) hepatotoxicity estimated by plasma glutamic pyruvic transaminase (GPT) or plasma bilirubin responses followed a strict Hammett-type dependence on the electronic properties of the substituent, toxicity increasing dramatically with increasing electron donation (p = -3.4 and - 1.4, respectively). This observation is consistent with the possible involvement of a critical S-oxidative biotransformation in the production of hepatic injury by these compounds. p-Hydroxythiobenzamide was not hepatotoxic at the highest dose studied, possibly because routes of biotransformation other than S-oxidation (e.g., conjugation) supervened. 2,6-Disubstituted thiobenzamides bearing either electron-donating or electron-withdrawing substituents also failed to produce hepatic injury. This was suggested to result from the steric effect of the substituents forcing the thioamide group out of conjugation with the aromatic ring. TBSO was significantly more hepatotoxic than TB, as indicated by earlier and/or larger increases in plasma GPT, plasma bilirubin, and hepatic triglycerides. The hepatotoxicity of TBSO was partially blocked by SKF-525A or Noctylimidazole, but was not affected by pretreatment of the rats with phenobarbital. For para-substituted TBSO derivatives (p-XC,H,C(SO)NHZ; X = CH,, H, Cl) the slope of the Hammett correlation for the plasma GPT response was essentially identical to that for the parent TB derivatives. In contrast, the plasma bilirubin response to TBSO was not altered by substituents. The above findings show that the hepatotoxicity of TB derivatives depends strongly on chemical factors in a manner consistent with S-oxidation as a critical step leading to liver injury. However neither the toxic responses to TB nor those to TBSO are uniformly affected in a simple direct manner by induction or inhibition of cytochrome P-450 enzymes. This may indicate that other enzyme systems, oxidative, reductive, or both, may also play a role in governing the toxic responses to TB and TBSO.
We have previously reported that thiobenzamide, C6H,CSNH2, produces dose’ Supported in part by Biomedical Research Grant RR-5606 from the National Institutes of and by the University of Kansas General Research ’ Address correspondence to this author at partment of Medicinal Chemistry, University of Lawrence, Kans. 66045. 0041.008W80/110260-13$02.00/O Copyright 0 1960 by Academrc Press. Inc. AI1 rights of reproduction in any form reserved.
related liver injury in rats as revealed by increases in plasma bilirubin, plasma glutamic pyruvic transaminase, and hepatic triglycerides (Hanzlik et al., 1978). Thiobenzamide was more toxic in rats pretreated with phenobarbital, and its toxicity was diminished by SKF-525A treatment, suggesting that its toxicity was dependent on
Support Health, Fund. the DeKansas,
260
THIOBENZAMIDE
HEPATOTOXICITY
261
N-Octylimidazole was synthesized by heating the cormetabolic activation. Compared to thiobenzamide, p-methoxythiobenzamide was a responding alkyl bromide with imidazole at 100°C for as described by Wilkinson et al., (1974). much more potent hepatotoxin and p- 3 hr The thiobenzamides and their S-oxide derivatives chlorothiobenzamide was much less so. This were administered intraperitoneally as very fine sussequence of relative toxicity might be the pensions in corn oil (4 ml/kg). Other drug treatments result of electronic substituent effects on were as follows: phenobarbital, 60 mg/kg, ip, once either the relative rates of obligatory meta- daily for 3 days prior to the experiment; SKF-525A 40 mgikg, ip, 30 min prior to an experiment; Nbolic activation steps, or the relative reac- octylimidazole. two 10 mg/kg doses, ip, as its HCI salt tivities of toxic metabolites. One type of in water, 30 min prior to and 30 min after thiometabolite expected for these compounds is benzamide-S-oxide treatment. For preparation of tissue sections, the tissue was the corresponding benzonitrile, but these rapidly excised after sacrifice and placed in 10% were shown not to be hepatotoxic. Another formalin for fixation. The tissues were then embedded type of anticipated metabolite is the S-oxide in paraffin, sectioned, and stained with hematoxylin or sulfine derivative, CGH,C( SO)NH,. In and eosin for light microscopic examination. this communication we report on the Student’s t test was used for statistical evaluation of the difference between two means. Multiple means hepatotoxicity of thiobenzamide-S-oxide vis-a-vis that of thiobenzamide, and on the were analyzed by a randomized one-way analysis of When the analysis indicated that a sigeffects of inducers and inhibitors of drug variance. nificant difference existed, the means of each group metabolizing enzymes on the toxicity of were compared by the Student-Newman-Keuls test thiobenzamide-S-oxide. We also char- (Sokal and Rohlf, 1%9). In all analyses the level of acterize in detail the effects of ring sub- significance chosen wasp < 0.05. stituents on the hepatotoxicity of thiobenzamide (TB) and thiobenzamide-S-oxide RESULTS (TBSO).
METHODS Male Sprague-Dawley rats (180-220 g) obtained from ARS/Sprague-Dawley, Madison, Wisconsin, were used throughout the study. They were maintained in a temperature controlled room with 12-hr periods of light and darkness, and had continuous access to tap water and Purina chow. The methods for determination of hepatic triglycerides, plasma glutamic pyruvic transaminase (GPT), and plasma bilirubin were the same as described previously (Hanzlik et al., 1978). Thiobenzamide was purchased from Aldrich Chemical Company; the other thiobenzamides were synthesized in our laboratory according to the method of Fairful er al. (1952). The thiobenzamide-S-oxide derivatives were synthesized by dissolving the thioamide in a minimum volume of methanol at 0°C followed by slow addition of a slight excess of 30% hydrogen peroxide. Pure S-oxides were isolated by evaporation of the methanol at room temperature on a rotary evaporator, dry column chromatography, and recrystallization from acetonitrile. All compounds were thoroughly characterized by appropriate spectra1 and analytical methods and were greater than 9% pure; the details of this will be published elsewhere.
Our earlier studies with thiobenzamide and two of its derivatives (p-chloro- and pmethoxy-) suggested that the relative hepatotoxicities of these compounds depended on the electronic character of the para-substituent group, being increased by the electron donating p-methoxy group and decreased by the electron-withdrawing pchloro substituent. To investigate this aspect more thoroughly, a series of five thiobenzamides (p-XC,H,CSNH,, where X = CH30, CH3, H, Cl, or CF,) was prepared and evaluated using the plasma bilirubin and plasma GPT responses to assess their relative hepatotoxicities. In this study it was found necessary to use two dosage levels with the different compounds because 2.0 mmol/kg of the more toxic compounds (p-CH,O, p-CH3) was lethal, while 0.8 mmoVkg of the less toxic compounds (pCl, p-CF,) did not produce responses significantly above control values. Plasma bilirubin and GPT responses were measured 24
262
HANZLIK,
CASHMAN,
k ” 2.5e 4 P 0 2.0s
-0.2
0
0.2
0.4
0.6
ob
FIG. 1. Hammett plot of the plasma glutamic pyruvic transaminase (GPT) response (units/ml) 24 hr after the administration of various puma-substituted thiobenzamides. Values are the mean t SE for three to six rats. Closed circles. 0.8 mmol/kg: open triangles, 2.0 mmolikg. The slope of the correlation is -3.42.
hr after ip administration of the thiobenzamide derivatives because earlier work with thiobenzamide (Vyas, 1979) had shown these responses to be maximal at this time. These results are shown in the form of Hammett plots in Figs. 1 and 2. In these plots (T is a parameter which reflects the electron-donating ((T < 0) or electron-withdrawing ((T > 0) properties of the parusubstituent (Lowry and Richardson, 1976). Both of these plots indicate an extremely good correlation between the toxic responses and the chemical properties of the thiobenzamide derivative as modulated by the electronic effects of the para-substituent. As assessed by both plasma GPT and plasma bilirubin, the toxicity of the thiobenzamides increases with increasing electron donation by the para-substituents. Such a pattern could, for example, be consistent with enzymatic S-oxidation as an obligatory bioactivation step, as has been suggested for thioacetamide (Hunter et al.,
AND
TRAIGER
1977; Porter et al., 1979; Porter and Neal, 1978), carbon disulfide (De Matteis and Seawright, 1973; Dalvi et al., 1974; De Matteis. 1974), and various thiourea derivatives (De Matteis, 1974; Boyd and Neal, 1976). In contrast, no correlation was found between toxicity of thep-substituted thiobenzamides and the Hansch subsfituent parameter, n, which characterizes the effects of substituents on the lipid/water partitioning behavior of the molecule (Hanschef al., 1973). Not all thiobenzamides tested conform to the structure-toxicity correlation found above. For example, during these studies phydroxythiobenzamide was prepared and found to be devoid of hepatotoxicity in doses as high as 2.0 mmol/kg. This is unexpected, considering the strongly electron donating character of the p-hydroxy group (a,, = -0.37). It is possible that this compound is metabolized in \,ilw quite differently from the others, perhaps by rapid conjugation processes such as 0-sulfation or 0-glucuronidation. Scheline et al. (1961) noted that introduction of a p-hydroxy group into the lung toxin phenylthiourea greatly diminished its toxicity and shifted its metabolism largely (80%) to pathways not involving “oxidative desulfurization,” i.e., glucuronidation and sulfation. The herbicide 2,6-dichlorothiobenzamide
‘72.0-’ \ I z 28 g 1.5 -I ------_ + - - - - _ _ ~Controls N \ 1.0
OCH,CH, I 1 1
-0.2
H I
0
Cl I I
0.2
FIG. 2. Hammett plot of the sponse (mgi100 ml plasma) 24 hr tion of various thiobenzamides. + SE for three to six rats. Closed open triangles 2.0 mmot/kg. The tion is - 1.40.
C$ 1
1
0.4
0.6
plasma bilirubin reafter the administraValues are the mean circles, 0.8 mmol/kg; slope of the correla-
THIOBENZAMIDE TABLE EFFECT
1
OF 2,6-~ISUBSTITUTED THIOBENZAMIDES PLASMA BILIRUBIN AND GPT ACTIVITY
ON
GFT Bilirubin (mgilO0 ml)
Treatment” 2,6-Dichlorothiobenzamide 2-Chloro-6-methylthiobenzamide 2,6-Dimethylthiobenzamide Control
263
HEPATOTOXICITY
activity (units/ml)
0.34 2 0.04
23 f 3
0.19 2 0.01
13 + 2
0.22 -+ 0.01 0.18 t 0.01
18 2 2 27 t 3
‘I Compounds were administered ip at a dosage of 1.0 mmol/kg, and animals were sacrificed 24 hr later. Valuesgivenare the means + SEforgroupsofthreerats.
(Prefix) is reported not to be hepatotoxic in rats (Griffiths ef al., 1966). This apparent lack of toxicity could perhaps result from the electronic substituent effect of the two chlorines, but it might also result from their steric effect, since they force the thioamide group into a plane essentially orthogonal to the plane of the phenyl ring, thereby elimi-
nating the possibility of conjugation or resonance interaction between the aromatic system and the thioamide group. Although this could potentially affect the oxidative metabolism of the thioamide moiety, the major metabolite of Prefix is 2,6-dichlorobenzonitrile (Griffiths et al., 1966), which most likely arises via an S-oxidative process as suggested earlier (Hanzlik ef al., 1978). In order to help resolve this issue we prepared and tested the thiobenzamides listed in Table 1. These compounds were administered ip at a dosage of 1.O mmol/kg and the plasma bilirubin and GPT responses were determined 24 hr later. Since 2,6disubstituted thiobenzamides bearing either electron-donating or electron-withdrawing groups were found to be equally nontoxic, the substituent effects in this case must be due to steric factors rather than electronic effects of the kind observed for the parasubstituted thiobenzamides. Further experimentation will be required to resolve this point definitively. Because of their likely involvement in the production of liver injury by thiobenzamide
600
500
2 ‘, .z 1
400
300
t ” 200
100
Time (hours1
FIG. 3. Time course for the plasma GPT response (units/ml) to 1.2 mmoVkg thiobenzamide thiobenzamide-S-oxide given ip in corn oil. Values are the mean -C SE for groups of four rats. asterisk indicates significant differences between the treated groups (p < 0.05).
or An
264
HANZLIK,
CASHMAN,
AND
TRAIGER
1.2 t
‘;i 5 9 a e
1.0 -
O-8-
g m r g
0.6 -
g m
0.4 -
0.2 -
I
I
6
12
I
1
I
24
46
72
Time (hours)
FIG.
benzamide indicates
4. Time course for the plasma bilirubin response (mgi100 ml plasma) to 1.2 mmohkg thioor thiobenzamide-S-oxide. Values are the mean f SE for groups of four rats. An asterisk significant differences between the treated groups (p < 0.05).
derivatives, we synthesized and evaluated the hepatotoxicity of thiobenzamide-soxide (TBSO) and two of its derivatives (p-CH3 and p-Cl). Preliminary results with TBSO suggested that it had a very steep dose-response relationship for toxicity; a dosage of 0.8 mmoYkg, ip, caused only small
rises in plasma GPT and bilirubin, while 1.5 mmol/kg was generally lethal within 12- 18 hr. A dosage of 1.2 mmol/kg, ip, was selected and used to compare the time courses for liver injury due to both TB and TBSO. Figures 3, 4, and 5 show the relative effects of these two compounds on
2
I
1
6
12
I
L
1
24
48
72
Time (hours)
FIG. 5. Time course for the hepatic triglyceride accumulation response (m&g liver) to 1.2 mmol/kg thiobenzamide or thiobenzamide-S-oxide. Values are the mean + SE for groups offour rats. An asterisk indicates significant differences between the treated groups (p < 0.05).
THIOBENZAMIDE
plasma GPT, plasma bilirubin, and hepatic triglycerides, respectively. As seen in Fig. 4, the plasma bilirubin response to TBSO is significantly higher at 6, 12, and 24 hr after treatment than that due to TB. Similarly the plasma GPT response at 12 hr and hepatic triglyceride response at 24 hr are significantly higher for TBSO than for TB. These biochemical indications of hepatotoxicity were confirmed by histological examination of liver sections taken 24 hr after treatment with either TB or TBSO (Figs. 6 and 7, respectively). In both cases a characteristic pattern of centrolobular
FIG. 6. Liver section from a rat 24 hr after toxylin and eosin stain: x90.
265
HEPATOTOXICITY
treatment
necrosis is readily apparent. Thus, in general, the hepatotoxic response to TBSO is qualitatively similar to but more rapid and/or intense than that due to TB. Our earlier study of the effects of phenobarbital pretreatment and SKF-525A also suggested a role for oxidative biotransformation in the production of liver injury by thiobenzamide. We therefore investigated the effect of phenobarbital, SKF525A, and N-octylimidazole, a very potent inhibitor of cytochrome P-450 (Wilkinson et al., 1974). on the hepatotoxicity of TBSO. These results, which are given in
with thiobenzamide
(1.2 mmol/kg,
ip). Hema-
266
HANZLIK,
CASHMAN,
AND TRAIGER
FIG. 7. Liver section from a rat 24 hr after treatment with thiobenzamide-S-oxide Hematoxylin and eosin stain; x90.
Table 2, show that while the toxicity of TBSO is in general diminished by SKF525A and N-octylimidazole, it is not enhanced by phenobarbital pretreatment. Thus, as was found for thiobenzamide, an effect of metabolism on the toxicity of TBSO is suggested, but not clearly revealed, through this approach. As a final probe of the possible requirement for further metabolic activation of the S-oxides, the effect of para-substituents on the hepatotoxicity of TBSO was investigated. As seen in Fig. 8, the plasma GFT response shows a good correlation with substituent electronic character, the p value
(I .2 mmol/kg, ip).
of -3.23 being nearly identical to that for the parent thiobenzamide series (Fig. 1). On the other hand, Fig. 9 shows that the plasma bilirubin response to the S-oxides is essentially independent of the substituents (p 2 -0.2), although this response to the parent thiobenzamides does vary according to the substituent electronic character (Fig. 2).
DISCUSSION Thiobenzamide has previously been shown to produce dose-related liver injury
THIOBENZAMIDE
TABLE EFFECT
267
HEPATOTOXICITY 2
OF SKF-RSA, N-OCTYLIMIDAZOLE, THIOBENZAMIDE-S-OXIDE-INDUCED
AND PHENOBARBITAL HEPATOTOXICITY~
ON
n
GPT (units/ml)
Bilirubin (mg/lOO ml)
Triglyceride (mg/g liver)
Control Thiobenzamide-S-oxide SKF-525A + thiobenzamide-S-oxide N-Octylimidazole + thiobenzamide-S-oxide
6 IO 5 5
282 506 2 113 4 101 ?
0.22 0.88 0.50 0.30
6.1 13.18 5.78 7.2
Thiobenzamide-S-oxide Phenobarbital + thiobenzamide-S-oxide
5 5
306 + 94 235 2 34
Treatment
2 82 25* 24h
+ 2 2 lr
0.01 0.13 0.10 0.02”
0.59 k 0.15 0.43 t 0.06
sz 0.3 2 0.9 -+ 1.0* r 0.5D
10.3 t 0.8 15.8 -e 2.4
a Rats were pretreated with phenobarbital (60 mgikg, ip) for 3 days. Thiobenzamide-S-oxide (1.2 mmolikg, 184 mg/kg, ip) was administered 24 hr after the last phenobarbital injection. SKF-525A (40 mgkg, ip) was administered 30 min prior to thiobenzamide-.S-oxide. A total of 20 mg/kg N-octylimidazole was administered ip 30 min prior and 30 min after thiobenzamide-S-oxide. * Statistically different from thiobenzamide-S-oxide (p < 0.05).
as indicated by increases in plasma concentrations of GPT and bilirubin, and hepatic triglyceride content (Hanzlik et al., 1978). It also produces acute histopathological changes in liver very similar to those induced by thioacetamide, i.e., depletion of glycogen in centrolobular hepatocytes as early as 6 hr after dosing progressing to
FIG. 8. Hammett plot of the plasma GPT response (units/ml) 24 hr after ip administration of 0.8 mmoYkg of various p-substituted thiobenzamide-S-oxides. Values are the mean ? SE for four rats. The slope of the correlation is -3.23.
severe centrolobular necrosis by 24 hr (Malvaldi, 1977; Chieli et al., 1979). From our initial work it appeared that the toxicity of thiobenzamide derivatives could be increased or decreased by para substituents which were electron-donating or -withdrawing, respectively. This was particularly intriguing because it suggested a link between the chemical properties of the thiobenzamides (or metabolites thereof) and the production of tissue injury. In particular the increase in toxicity by electron donating substituents would be understandable if oxidative biotransformation of thiobenzamide and its derivatives were required for production of toxicity in rive, as has been suggested for other thiocarbonyl compounds including thioacetamide (Hunter et al., 1977; Porter et al., 1979; Porter and Neal, 1978), carbon disulfide (De Matteis and Seawright, 1973; Dalvi et al., 1974; De Matteis, 1974), and various thiourea derivatives (De Matteis, 1974; Boyd and Neal, 1976). To investigate this aspect more thoroughly we have now determined the relative hepatotoxicity of five para-substituted thiobenzamides using increases in plasma GPT and bilirubin concentrations to assess liver injury. The results of this study appear
268
HANZLIK,
CASHMAN,
as Hammett plots in Figs. 1 and 2. Several features of these plots are particularly worthy of note. The excellent linear correlations between the toxic response and the Hammett sigma parameter, which characterizes the electron-donating or -withdrawing ability of the pnra-substituent (Lowry and Richardson, 1976), indicates that the relative toxicities of these compounds are indeed strongly dependent on their chemicat properties. Since no correlation was found between toxicity and the Hansch 7r parameter, which reflects the effects of the substituents on the lipid/water partitioning behavior of the molecules (Hansch et al.. 1973), it is most unlikely that the observed differences in toxicity result from differences in the absorption and/or distribution of the thiobenzamides. Rather, toxicity would appear to be linked to a chemical process such as oxidative biotransformation (vide i+.~). Furthermore. the fact that both groups of compounds at the two dosages used gave parallel Hammett plots (Figs. 1 and 2) strongly suggests that all five compounds share a common mechanism of action. The sign and magnitude of the slope of the Hammett correlation, i.e., the Hammett p value, also yield important information characterizing the chemical nature of the rate-limiting step in whatever sequence of events, as yet unknown. leads from the parent thioamide to the expression of toxicity in I~~~w. For the plasma GPT response the large negative p value of -3.42 suggests that some critical step leading to toxicity (1) involves the parent thiobenzamide or some closely relmted metabolite, and (2) involves an interaction which is very strongly facilitated by electron donation from ring substituents. In particular a large negative p value would be consistent with enzymatic S-oxidation as an obligatory step leading from thiobenzamide to the production of liver injury. Such p values have been observed in the chemical Soxidation of thiobenzophenones (Battaglia
AND
TRAIGER 2.0
FIG. (mg/lOO
1
9. Hammett plot ofthe plasma bilirubin response ml plasma)
24 hr after
ip administration
of
0.8 mmoVkg of various p-substituted thiobenzamideS-oxides. Values are the mean f SE for four rats. The slope of the line drawn is -0.2.
et al.,
1971). and thiobenzamide derivatives (J. R. Cashman, unpublished observations). Conversely, positive p values are observed for reactions involving nucleophilic attack on thiocarbonyl compounds (Drobnica and Augustin, 1965; Kardos et rd.. 1965), reflecting their facilitation by electron-withdrawing substituents. This point is especially significant in light of the importance usually ascribed to covalent binding of reactive metabolites (electrophiles) to celhilar macromolecules (nucleophiles) in cases of tissue injury due to chemicals (Jollow et ml., 1978). Many instances of tissue injury due to chemicals appear to result from oxidative biotransformation of the substance to a chemically reactive metabolite, often an electrophilic agent, which then alkylates various cellular nucleophiles leading uitimately to cell injury or death. In the present case the large negative rho values for the Hammett correlations in Figs. 1 and 2 should fzor be taken to imply that covalent binding of an electrophilic chemically reactive metabolite of thiobenzamide is not involved in the production of toxicity. They only imply that if such a covalent binding step is involved, it is not the rate-limiting step in the process leading from the thiobenzamide to the expression of toxicity. The failure of p-hydroxythiobenzamide to conform to the Hammett structure-
THIOBENZAMIDE DETOXICATION
BIOCHEMICAL
EVENTS
ALTERATIONS
DISTURBANCE
ASSESSMENT
AND RESPONSES;
OF HOMEOSTASIS
OF TOXICITY
FIG. 10. Hypothetical model relating the in viva disposition of TB and TBSO to the production of toxic responses. For details see discussion in text.
toxicity correlations presented in Figs. 1 and 2 can potentially be attributed to its ability to undergo rapid conjugation with glucuronic acid and/or sulfate, and thereby avoid Soxidative pathways leading to toxic metabolites, as mentioned above (Scheline et al., 1961). However, the lack of hepatotoxicity of 2,6-dichlorothiobenzamide is not likely to be due to a major shift in metabolism since its major metabolite, 2,6-dichlorobenzonitrile, most likely arises via S-oxidative metabolism. Nor can it be explained by the electron withdrawing effect of the two chlorines, since 2,6-dimethylthiobenzamide is equally nontoxic in the rat. The lack of toxicity of 2,6-disubstituted thiobenzamides can probably be attributed to (1) the ready decomposition of the S-oxides to the nitrile metabolite, which was previously shown not to be hepatotoxic in the rat (Griffiths et al., 1966), and/or (2) unfavorable steric effects of the substituents on a putative second S-oxidation step, as discussed
HEPATOTOXICITY
269
further below. Further studies in progress should help to clarify this point. Both the electronic substituent effects discussed above and earlier results with inducers and inhibitors of drug metabolism suggested the possible involvement of enzymatic S-oxidation of thiobenzamide in the expression of its toxicity. Further support for this hypothesis comes from comparison of the time courses of plasma GPT and bilirubin and hepatic triglyceride concentrations following equimolar ip doses of TB and TBSO (Figs. 3, 4, and 5). The faster onset and/or greater intensity of these responses to TBSO is generally consistent with its obligatory involvement in liver injury by TB. However, before attempting to analyze TBSO vis-a-vis TB in greater detail, it is useful to consider the diagram in Fig. 10. Following ip administration of TB it must be absorbed and transported to the liver (ki). AS it is absorbed it can begin to undergo biotransformation. Thiobenzamide is known to be S-oxidized by a purified microsomal flavin-containing monooxygenase (Ziegler et al., 1979), and in vitro studies with rat liver microsomes in our laboratory have shown that TB is rapidly and extensively converted to TBSO (J. R. Cashman, unpublished observations), thus establishing the existence of the k, step in Fig. 10. Alternatively, of course, TBSO may be administered directly (kf ). Back-reduction of TBSO to TB (k-,) has not been specifically investigated but similar reductions have been observed in vivo with related thioamide-S-oxides (Johnston et al., 1967; Ammon et al., 1967; Porter and Neal, 1978). Further oxidation of TBSO to TBSO, (kz) is also a likely possibility (Hunter et al., 1977; Porter ef al., 1979). Potentially TB, TBSO, and TBSO, can each be “detoxified” (kdr kh, k$, respectively) by a variety of processes such as biotransformation or elimination. Presumably TB or its metabolites also interact in various deleterious ways with critical cellular targets
270
HANZLIK,
CASHMAN,
such as enzymes or macromolecules, as represented by k,, k;, and k?. These latter events will perturb the normal homeostatic balance of the cell, and if the perturbation is large enough, it will be detected at some point by histological examination of the tissue and/or by measuring some biochemical parameter of cell function or integrity. Of course, it must be borne in mind that such attempts to assess toxicity are merely instantaneous glimpses of a complex dynamic process. In light of the above discussion one can now consider the “relative toxicities” of equimolar doses of TB and TBSO in terms of both the rate of onset and the ultimate intensity of tissue injury. If TBSO is an obligatory intermediate in toxic responses to TB, the actual response to its presence in the target cell will presumably depend on the balance between processes represented by k; and those represented by ki and k-,. Thus if some interaction of TBSO with the cell is very proximate to the measured index of toxicity one would certainly expect a more rapid response to TBSO than to TB. However, if the interaction of TBSO with the cell were many subsequent steps removed from the measured index of toxicity one might not detect much difference in onset of response between TBSO and TB, especially if the formation of TBSO from TB were faster than some of the subsequent steps leading to the measured index of toxicity (k, > ki). Finally if the processes represented by k; and k_, are relatively easily saturated, a rapid input of TBSO by direct administration is more likely to produce a greater response than a slower input from enzymatic oxidation of an equimolar amount of TB. The above model, while hypothetical, can help to rationalize numerous observations pertaining to hepatic injury by derivatives of TB and TBSO. For example, the faster onset and greater overall response of plasma bilirubin to TBSO, and the moderate but negative rho value for Fig. 2 are consistent
AND
TRAIGER
with an obligatory role for TBSO in interfering with biliary function leading to hyperbilirubinemia. The lack of substituent effects seen in Fig. 9 would logically result if TBSO derivatives did not require further oxidation in order to interfere with biliary function. The increase in plasma bilirubin due to TB has previously been shown to result from a decreased ability of the liver both to extract organic anions from plasma and to secrete them into bile (Vyas, 1979). Furthermore, the liver necrosis caused by TB does not per se result in hyperbilirubinemia, because doses of carbon tetrachloride producing even significantly greater increases in plasma GPT do not alter plasma bilirubin levels (Vyas, 1979). In contrast, it would appear that the plasma GPT response might require further oxidative metabolism of TBSO, perhaps to TBSO,. This could explain why the p values of Figs. 1 and 8 are essentially identical but much more negative than the p value for Fig. 2. i.e., if the second oxidation of TB, not the first, were rate limiting for the production of necrosis. Similar kinetic trends are seen in chemical oxidations of thioamides; the second oxidation step is always much slower than the first (Battaglia et al., 1971), and less reactive series of thioamides show much larger negative p values (i.e., greater dependence on electron donation from substituents) than the more reactive ones. That the GPT response to TBSO is slightly faster in onset but not significantly greater in overall intensity than that due to TB would also be consistent with an obligatory role for TBS02, since formation of the latter would be governed mainly by k, irrespective of whether TB or TBSO were administered. In some cases in which a toxic response is due to a metabolite of the parent drug, induction or inhibition of the required biotransformation leads to consistent and predictable alterations in the toxic responses. With agents such as bromobenzene, carbon tetrachloride, or acetaminophen such alterations in toxicity form a major part of the
THIOBENZAMIDE
evidence implicating a toxic metabolite (Jollow ef al., 1978). The toxic responses to TB and TBSO, however, are not altered in a consistent way by phenobarbital pretreatment, SKF-525A or NOI. One reason for this might simply be the greater complexity of events involved in the disposition of and biological response to TB and TBSO, e.g., the probable reversibility of the first oxidation of TB (which has no analogy in the simpler cases mentioned above), or the possible association of different toxic responses with different metabolites. Another important reason may be the involvement of the microsomal flavin-containing monooxygenase in the oxidation of TB (see above), and possibly of TBSO as well, because this enzyme does not respond to phenobarbital as do the cytochromes P-450, nor is it much inhibited by SKF-525A or NOI. Better understanding of the effects of inducers and inhibitors on the toxicity of TB and TBSO must await detailed investigation of their metabolism and disposition; such studies are presently under way in our laboratory. ACKNOWLEDGMENTS We are grateful to the National Institutes of Health (RR-5606) and the University of Kansas General Research Fund for their financial support of this work.
REFERENCES AMMON. R., BERNINGER, H.. HAAS, H. J.. AND LANDSBERG. I. (1967). Thioacetamide-sulfoxid, ein Stofwechselprodukt des Thioacetamids. Arzneim. Forsch. 17, 521-523. BATTAGLIA, A., DONDONI, A., GIORGIANNI, P., MACCAGNANI. G., AND MAZZANTI, G. (1971). Sulhnes. III. Kinetics of oxidation of thiobenzophenones with peroxybenzoic acid. J. Chem. Sot. (B), 1547- 1550. Bovo, M. R., ANo NEAL, R. A. (1976). Studies on the mechanism of toxicity and of development of tolerance to the pulmonary toxin a-napthylthiourea (ANTU). Drug Metab. Dispos. 4, 314-322. CHIELI, E., MALVALDI, G., AND TONGIANI, R. (1979). Early biochemical liver changes following thiobenzamide poisoning. Toxicology 13, IOI- 114.
HEPATOTOXICITY
271
DALVI, R. R., POORE, R. E., AND NEAL, R. A. (1974). Studies of the metabolism of carbon disulfide by rat liver microsomes. L$f Sci. 14, 1785- 17%. DE MATTEIS, F.. AND SEAWRIGHT, A. (1973). Oxidative metabolism of carbon disulphide by the rat. Effect of treatments which modify the liver toxicity of C&. Chem. -Biol. Interact. 7, 375-388. DE MATTEIS, F. (1974). Covalent binding of sulfur to microsomes and loss of cytochrome P-450 during the oxidative desulfurization of several chemicals. Mol. Phormacol. 10, 849-854. DROBNICA. L., AND AUGUSTIN, J. (l%S). Reactions of isothiocyanates with amino acids, peptides, and proteins. I. Kinetics of the reaction of aromatic isothiocyanates with glycine. Cot/. C:rch. ClrPtn. C0mm1tr2. 30, 99- 104. FAIRFUL. A. E. S., LOWE. J. L.. AND PEAK, D. A. ( 1952). A convenient reagent for the preparation of thioamides, and the thiohydrolysis of S-alkyl-isothiourea derivatives. J. Cltrm. Sot., 742-744. GRIFFITHS, M. H., Moss. J. A., ROSE, J. A., AND HATHWAY. D. E. (1966). The comparative metabolism of 2.6-dichlorothiobenzamide (Prefix) and 2,6-dichlorobenzonitrile in the dog and rat. Biochem. J. 98, 770-781. HANSCH. C., LEO. A., UNGER, S. H., KIM, K. H.. NIKAITANI. D., AND LIEN, E. J. (1973). Aromatic substituent constants for structure-activity correlations. J. Med. Chum. 16, 1207-1216. HANZLIK, R. P., VYAS. K. P., AND TRAIGER, G. J. (1978). Substituent effects on the hepatotoxicity of thiobenzamide derivatives in the rat. To.rico/. Appl. Phnrmacol. 46, 685-694. HUNTER, A. L.. HOLXHER, M. A., AND NEAL, R. A. ( 1977). Thioacetamide induced hepatic necrosis. I. Involvement of the mixed-function oxidase enzyme system. J. Pharmcrcol. Exp. Ther. 200, 439-448. JOHNSTON, J. P.. KANE, P. O., AND KIFGBY, M. R. (1967). The metabolism of ethionamide and its sulfoxide. .I. Phurm. Phnrmucol. 19, 1-9. JOLLOW, D. J., Kocsrs, J. J.. SNYDER, R.. AND VAINIO, H. (1978). Biological Reactive Intermedicttes. Plenum, New York. KAE~D~S, A. M., VOLKE, J., AND KRISTIAN. P. (1965). Polarography of phenyl isothiocyanates substituted in meta-position and kinetics of their reactions with hydroxyl ions. Co/l. Czech. Chem. Commun. 30, 931-938. LOWRY, R. H., AND RICHARDSON, K. S. (1976). Mechanism and Theory in Organic Chemistry, pp. 60-71. Harper & Row, New York. MALVALDI. G. (1977). Liver changes following thiobenzamide poisoning. Experientia 33, 1200-1201. PORTER, W. R., AND NEAL, R. A. (1978). Metabolism of thioacetamide and thioacetamide-S-oxide by rat liver microsomes. Drug Metub. Dispos. 6,379-388. PORTER, W. R., GUDZINOWECZ. M. J., AND NEAL,
272
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CASHMAN,
R. A. (1979): Thioacetamide-induced hepatic necrosis. II. Pharmacokinetics of thioacetamide and thioacetamide-S-oxide in the rat. J. Pharmacol. Exp. Ther. 208, 386-391. SCHELINE, R. R.. SMITH, R. L.. AND WILLIAMS, R. T. (1961). The metabolism of arylthioureas. II. The metabolism of Y- and 35S-labeIed I-phenyl-2-thiourea and its derivatives. J. Med. Pharm. Chem. 4, 109135. SOKAL, R. R., AND ROHLF, F. J. (eds.) (1969). In Biometry, The Principles and Practice of Statistics in Biological Research, pp. 239-246. Freeman, San Francisco, Calif.
AND TRAIGER
VYAS. K. P. (1979).
of Thiocarbonyl
Metabolism Compounds.
and Hepatotoxicity
Ph.D. Thesis, Univer-
sity of Kansas. WILKINSON, C. F., HETNARSKI, K., CANTWELL, G. P., AND DICARLO, F. J. (1974). Structure-activity relationships in the effects of I-alkylimidazoles on microsomal oxidation in vitro and in Go. Biochem. Pharmacol.
23, 2377-2386.
ZIEGLER, D. M., POULSEN, L. L., AND DUFFEL, M. W. (1979). Kinetic mechanism and substrate specificity of the microsomal FAD-containing monooxygenase. In 4th Internat. Symposium on Microsomes and Drug Oxidations, Ann Arbor, Mich., abstract L-303.
AND APPLIED
Relative
PHARMACOLOGY
(1980)
Hepatotoxicity of Substituted Thiobenzamides Thiobenzamide-S-oxides in the Rat1
ROBERT P. HANZLIK,~ Department
55, 260-272
of Medicinal
Chemistry,
Received
JOHN
R.
CASHMAN,
of Pharmacology Kansas 66045
and Department Lawrence.
November
12, 1979; accepted
AND
GEORGE and Toxicology,
and
J. TRAIGER University
of Kansas.
June 2, 1980
Relative Hepatotoxicity of Substituted Thiobenzamides and Thiobenzamide-S-oxides in the Rat. HANZLIK, R. P., CASHMAN, J. R., AND TRAIGER, G. J.(1980). Toxicol. Appl. Pharmacol. 55, 260-272. The effect of substituents on the relative hepatotoxicity of various derivatives of thiobenzamide (TB) and thiobenzamide-S-oxide (TBSO) has been investigated in the rat. For five para-substituted thiobenzamides (p-XCGH,CSNH,: X = CH,O, CH,, H, Cl, CF,) hepatotoxicity estimated by plasma glutamic pyruvic transaminase (GPT) or plasma bilirubin responses followed a strict Hammett-type dependence on the electronic properties of the substituent, toxicity increasing dramatically with increasing electron donation (p = -3.4 and - 1.4, respectively). This observation is consistent with the possible involvement of a critical S-oxidative biotransformation in the production of hepatic injury by these compounds. p-Hydroxythiobenzamide was not hepatotoxic at the highest dose studied, possibly because routes of biotransformation other than S-oxidation (e.g., conjugation) supervened. 2,6-Disubstituted thiobenzamides bearing either electron-donating or electron-withdrawing substituents also failed to produce hepatic injury. This was suggested to result from the steric effect of the substituents forcing the thioamide group out of conjugation with the aromatic ring. TBSO was significantly more hepatotoxic than TB, as indicated by earlier and/or larger increases in plasma GPT, plasma bilirubin, and hepatic triglycerides. The hepatotoxicity of TBSO was partially blocked by SKF-525A or Noctylimidazole, but was not affected by pretreatment of the rats with phenobarbital. For para-substituted TBSO derivatives (p-XC,H,C(SO)NHZ; X = CH,, H, Cl) the slope of the Hammett correlation for the plasma GPT response was essentially identical to that for the parent TB derivatives. In contrast, the plasma bilirubin response to TBSO was not altered by substituents. The above findings show that the hepatotoxicity of TB derivatives depends strongly on chemical factors in a manner consistent with S-oxidation as a critical step leading to liver injury. However neither the toxic responses to TB nor those to TBSO are uniformly affected in a simple direct manner by induction or inhibition of cytochrome P-450 enzymes. This may indicate that other enzyme systems, oxidative, reductive, or both, may also play a role in governing the toxic responses to TB and TBSO.
We have previously reported that thiobenzamide, C6H,CSNH2, produces dose’ Supported in part by Biomedical Research Grant RR-5606 from the National Institutes of and by the University of Kansas General Research ’ Address correspondence to this author at partment of Medicinal Chemistry, University of Lawrence, Kans. 66045. 0041.008W80/110260-13$02.00/O Copyright 0 1960 by Academrc Press. Inc. AI1 rights of reproduction in any form reserved.
related liver injury in rats as revealed by increases in plasma bilirubin, plasma glutamic pyruvic transaminase, and hepatic triglycerides (Hanzlik et al., 1978). Thiobenzamide was more toxic in rats pretreated with phenobarbital, and its toxicity was diminished by SKF-525A treatment, suggesting that its toxicity was dependent on
Support Health, Fund. the DeKansas,
260
THIOBENZAMIDE
HEPATOTOXICITY
261
N-Octylimidazole was synthesized by heating the cormetabolic activation. Compared to thiobenzamide, p-methoxythiobenzamide was a responding alkyl bromide with imidazole at 100°C for as described by Wilkinson et al., (1974). much more potent hepatotoxin and p- 3 hr The thiobenzamides and their S-oxide derivatives chlorothiobenzamide was much less so. This were administered intraperitoneally as very fine sussequence of relative toxicity might be the pensions in corn oil (4 ml/kg). Other drug treatments result of electronic substituent effects on were as follows: phenobarbital, 60 mg/kg, ip, once either the relative rates of obligatory meta- daily for 3 days prior to the experiment; SKF-525A 40 mgikg, ip, 30 min prior to an experiment; Nbolic activation steps, or the relative reac- octylimidazole. two 10 mg/kg doses, ip, as its HCI salt tivities of toxic metabolites. One type of in water, 30 min prior to and 30 min after thiometabolite expected for these compounds is benzamide-S-oxide treatment. For preparation of tissue sections, the tissue was the corresponding benzonitrile, but these rapidly excised after sacrifice and placed in 10% were shown not to be hepatotoxic. Another formalin for fixation. The tissues were then embedded type of anticipated metabolite is the S-oxide in paraffin, sectioned, and stained with hematoxylin or sulfine derivative, CGH,C( SO)NH,. In and eosin for light microscopic examination. this communication we report on the Student’s t test was used for statistical evaluation of the difference between two means. Multiple means hepatotoxicity of thiobenzamide-S-oxide vis-a-vis that of thiobenzamide, and on the were analyzed by a randomized one-way analysis of When the analysis indicated that a sigeffects of inducers and inhibitors of drug variance. nificant difference existed, the means of each group metabolizing enzymes on the toxicity of were compared by the Student-Newman-Keuls test thiobenzamide-S-oxide. We also char- (Sokal and Rohlf, 1%9). In all analyses the level of acterize in detail the effects of ring sub- significance chosen wasp < 0.05. stituents on the hepatotoxicity of thiobenzamide (TB) and thiobenzamide-S-oxide RESULTS (TBSO).
METHODS Male Sprague-Dawley rats (180-220 g) obtained from ARS/Sprague-Dawley, Madison, Wisconsin, were used throughout the study. They were maintained in a temperature controlled room with 12-hr periods of light and darkness, and had continuous access to tap water and Purina chow. The methods for determination of hepatic triglycerides, plasma glutamic pyruvic transaminase (GPT), and plasma bilirubin were the same as described previously (Hanzlik et al., 1978). Thiobenzamide was purchased from Aldrich Chemical Company; the other thiobenzamides were synthesized in our laboratory according to the method of Fairful er al. (1952). The thiobenzamide-S-oxide derivatives were synthesized by dissolving the thioamide in a minimum volume of methanol at 0°C followed by slow addition of a slight excess of 30% hydrogen peroxide. Pure S-oxides were isolated by evaporation of the methanol at room temperature on a rotary evaporator, dry column chromatography, and recrystallization from acetonitrile. All compounds were thoroughly characterized by appropriate spectra1 and analytical methods and were greater than 9% pure; the details of this will be published elsewhere.
Our earlier studies with thiobenzamide and two of its derivatives (p-chloro- and pmethoxy-) suggested that the relative hepatotoxicities of these compounds depended on the electronic character of the para-substituent group, being increased by the electron donating p-methoxy group and decreased by the electron-withdrawing pchloro substituent. To investigate this aspect more thoroughly, a series of five thiobenzamides (p-XC,H,CSNH,, where X = CH30, CH3, H, Cl, or CF,) was prepared and evaluated using the plasma bilirubin and plasma GPT responses to assess their relative hepatotoxicities. In this study it was found necessary to use two dosage levels with the different compounds because 2.0 mmol/kg of the more toxic compounds (p-CH,O, p-CH3) was lethal, while 0.8 mmoVkg of the less toxic compounds (pCl, p-CF,) did not produce responses significantly above control values. Plasma bilirubin and GPT responses were measured 24
262
HANZLIK,
CASHMAN,
k ” 2.5e 4 P 0 2.0s
-0.2
0
0.2
0.4
0.6
ob
FIG. 1. Hammett plot of the plasma glutamic pyruvic transaminase (GPT) response (units/ml) 24 hr after the administration of various puma-substituted thiobenzamides. Values are the mean t SE for three to six rats. Closed circles. 0.8 mmol/kg: open triangles, 2.0 mmolikg. The slope of the correlation is -3.42.
hr after ip administration of the thiobenzamide derivatives because earlier work with thiobenzamide (Vyas, 1979) had shown these responses to be maximal at this time. These results are shown in the form of Hammett plots in Figs. 1 and 2. In these plots (T is a parameter which reflects the electron-donating ((T < 0) or electron-withdrawing ((T > 0) properties of the parusubstituent (Lowry and Richardson, 1976). Both of these plots indicate an extremely good correlation between the toxic responses and the chemical properties of the thiobenzamide derivative as modulated by the electronic effects of the para-substituent. As assessed by both plasma GPT and plasma bilirubin, the toxicity of the thiobenzamides increases with increasing electron donation by the para-substituents. Such a pattern could, for example, be consistent with enzymatic S-oxidation as an obligatory bioactivation step, as has been suggested for thioacetamide (Hunter et al.,
AND
TRAIGER
1977; Porter et al., 1979; Porter and Neal, 1978), carbon disulfide (De Matteis and Seawright, 1973; Dalvi et al., 1974; De Matteis. 1974), and various thiourea derivatives (De Matteis, 1974; Boyd and Neal, 1976). In contrast, no correlation was found between toxicity of thep-substituted thiobenzamides and the Hansch subsfituent parameter, n, which characterizes the effects of substituents on the lipid/water partitioning behavior of the molecule (Hanschef al., 1973). Not all thiobenzamides tested conform to the structure-toxicity correlation found above. For example, during these studies phydroxythiobenzamide was prepared and found to be devoid of hepatotoxicity in doses as high as 2.0 mmol/kg. This is unexpected, considering the strongly electron donating character of the p-hydroxy group (a,, = -0.37). It is possible that this compound is metabolized in \,ilw quite differently from the others, perhaps by rapid conjugation processes such as 0-sulfation or 0-glucuronidation. Scheline et al. (1961) noted that introduction of a p-hydroxy group into the lung toxin phenylthiourea greatly diminished its toxicity and shifted its metabolism largely (80%) to pathways not involving “oxidative desulfurization,” i.e., glucuronidation and sulfation. The herbicide 2,6-dichlorothiobenzamide
‘72.0-’ \ I z 28 g 1.5 -I ------_ + - - - - _ _ ~Controls N \ 1.0
OCH,CH, I 1 1
-0.2
H I
0
Cl I I
0.2
FIG. 2. Hammett plot of the sponse (mgi100 ml plasma) 24 hr tion of various thiobenzamides. + SE for three to six rats. Closed open triangles 2.0 mmot/kg. The tion is - 1.40.
C$ 1
1
0.4
0.6
plasma bilirubin reafter the administraValues are the mean circles, 0.8 mmol/kg; slope of the correla-
THIOBENZAMIDE TABLE EFFECT
1
OF 2,6-~ISUBSTITUTED THIOBENZAMIDES PLASMA BILIRUBIN AND GPT ACTIVITY
ON
GFT Bilirubin (mgilO0 ml)
Treatment” 2,6-Dichlorothiobenzamide 2-Chloro-6-methylthiobenzamide 2,6-Dimethylthiobenzamide Control
263
HEPATOTOXICITY
activity (units/ml)
0.34 2 0.04
23 f 3
0.19 2 0.01
13 + 2
0.22 -+ 0.01 0.18 t 0.01
18 2 2 27 t 3
‘I Compounds were administered ip at a dosage of 1.0 mmol/kg, and animals were sacrificed 24 hr later. Valuesgivenare the means + SEforgroupsofthreerats.
(Prefix) is reported not to be hepatotoxic in rats (Griffiths ef al., 1966). This apparent lack of toxicity could perhaps result from the electronic substituent effect of the two chlorines, but it might also result from their steric effect, since they force the thioamide group into a plane essentially orthogonal to the plane of the phenyl ring, thereby elimi-
nating the possibility of conjugation or resonance interaction between the aromatic system and the thioamide group. Although this could potentially affect the oxidative metabolism of the thioamide moiety, the major metabolite of Prefix is 2,6-dichlorobenzonitrile (Griffiths et al., 1966), which most likely arises via an S-oxidative process as suggested earlier (Hanzlik ef al., 1978). In order to help resolve this issue we prepared and tested the thiobenzamides listed in Table 1. These compounds were administered ip at a dosage of 1.O mmol/kg and the plasma bilirubin and GPT responses were determined 24 hr later. Since 2,6disubstituted thiobenzamides bearing either electron-donating or electron-withdrawing groups were found to be equally nontoxic, the substituent effects in this case must be due to steric factors rather than electronic effects of the kind observed for the parasubstituted thiobenzamides. Further experimentation will be required to resolve this point definitively. Because of their likely involvement in the production of liver injury by thiobenzamide
600
500
2 ‘, .z 1
400
300
t ” 200
100
Time (hours1
FIG. 3. Time course for the plasma GPT response (units/ml) to 1.2 mmoVkg thiobenzamide thiobenzamide-S-oxide given ip in corn oil. Values are the mean -C SE for groups of four rats. asterisk indicates significant differences between the treated groups (p < 0.05).
or An
264
HANZLIK,
CASHMAN,
AND
TRAIGER
1.2 t
‘;i 5 9 a e
1.0 -
O-8-
g m r g
0.6 -
g m
0.4 -
0.2 -
I
I
6
12
I
1
I
24
46
72
Time (hours)
FIG.
benzamide indicates
4. Time course for the plasma bilirubin response (mgi100 ml plasma) to 1.2 mmohkg thioor thiobenzamide-S-oxide. Values are the mean f SE for groups of four rats. An asterisk significant differences between the treated groups (p < 0.05).
derivatives, we synthesized and evaluated the hepatotoxicity of thiobenzamide-soxide (TBSO) and two of its derivatives (p-CH3 and p-Cl). Preliminary results with TBSO suggested that it had a very steep dose-response relationship for toxicity; a dosage of 0.8 mmoYkg, ip, caused only small
rises in plasma GPT and bilirubin, while 1.5 mmol/kg was generally lethal within 12- 18 hr. A dosage of 1.2 mmol/kg, ip, was selected and used to compare the time courses for liver injury due to both TB and TBSO. Figures 3, 4, and 5 show the relative effects of these two compounds on
2
I
1
6
12
I
L
1
24
48
72
Time (hours)
FIG. 5. Time course for the hepatic triglyceride accumulation response (m&g liver) to 1.2 mmol/kg thiobenzamide or thiobenzamide-S-oxide. Values are the mean + SE for groups offour rats. An asterisk indicates significant differences between the treated groups (p < 0.05).
THIOBENZAMIDE
plasma GPT, plasma bilirubin, and hepatic triglycerides, respectively. As seen in Fig. 4, the plasma bilirubin response to TBSO is significantly higher at 6, 12, and 24 hr after treatment than that due to TB. Similarly the plasma GPT response at 12 hr and hepatic triglyceride response at 24 hr are significantly higher for TBSO than for TB. These biochemical indications of hepatotoxicity were confirmed by histological examination of liver sections taken 24 hr after treatment with either TB or TBSO (Figs. 6 and 7, respectively). In both cases a characteristic pattern of centrolobular
FIG. 6. Liver section from a rat 24 hr after toxylin and eosin stain: x90.
265
HEPATOTOXICITY
treatment
necrosis is readily apparent. Thus, in general, the hepatotoxic response to TBSO is qualitatively similar to but more rapid and/or intense than that due to TB. Our earlier study of the effects of phenobarbital pretreatment and SKF-525A also suggested a role for oxidative biotransformation in the production of liver injury by thiobenzamide. We therefore investigated the effect of phenobarbital, SKF525A, and N-octylimidazole, a very potent inhibitor of cytochrome P-450 (Wilkinson et al., 1974). on the hepatotoxicity of TBSO. These results, which are given in
with thiobenzamide
(1.2 mmol/kg,
ip). Hema-
266
HANZLIK,
CASHMAN,
AND TRAIGER
FIG. 7. Liver section from a rat 24 hr after treatment with thiobenzamide-S-oxide Hematoxylin and eosin stain; x90.
Table 2, show that while the toxicity of TBSO is in general diminished by SKF525A and N-octylimidazole, it is not enhanced by phenobarbital pretreatment. Thus, as was found for thiobenzamide, an effect of metabolism on the toxicity of TBSO is suggested, but not clearly revealed, through this approach. As a final probe of the possible requirement for further metabolic activation of the S-oxides, the effect of para-substituents on the hepatotoxicity of TBSO was investigated. As seen in Fig. 8, the plasma GFT response shows a good correlation with substituent electronic character, the p value
(I .2 mmol/kg, ip).
of -3.23 being nearly identical to that for the parent thiobenzamide series (Fig. 1). On the other hand, Fig. 9 shows that the plasma bilirubin response to the S-oxides is essentially independent of the substituents (p 2 -0.2), although this response to the parent thiobenzamides does vary according to the substituent electronic character (Fig. 2).
DISCUSSION Thiobenzamide has previously been shown to produce dose-related liver injury
THIOBENZAMIDE
TABLE EFFECT
267
HEPATOTOXICITY 2
OF SKF-RSA, N-OCTYLIMIDAZOLE, THIOBENZAMIDE-S-OXIDE-INDUCED
AND PHENOBARBITAL HEPATOTOXICITY~
ON
n
GPT (units/ml)
Bilirubin (mg/lOO ml)
Triglyceride (mg/g liver)
Control Thiobenzamide-S-oxide SKF-525A + thiobenzamide-S-oxide N-Octylimidazole + thiobenzamide-S-oxide
6 IO 5 5
282 506 2 113 4 101 ?
0.22 0.88 0.50 0.30
6.1 13.18 5.78 7.2
Thiobenzamide-S-oxide Phenobarbital + thiobenzamide-S-oxide
5 5
306 + 94 235 2 34
Treatment
2 82 25* 24h
+ 2 2 lr
0.01 0.13 0.10 0.02”
0.59 k 0.15 0.43 t 0.06
sz 0.3 2 0.9 -+ 1.0* r 0.5D
10.3 t 0.8 15.8 -e 2.4
a Rats were pretreated with phenobarbital (60 mgikg, ip) for 3 days. Thiobenzamide-S-oxide (1.2 mmolikg, 184 mg/kg, ip) was administered 24 hr after the last phenobarbital injection. SKF-525A (40 mgkg, ip) was administered 30 min prior to thiobenzamide-.S-oxide. A total of 20 mg/kg N-octylimidazole was administered ip 30 min prior and 30 min after thiobenzamide-S-oxide. * Statistically different from thiobenzamide-S-oxide (p < 0.05).
as indicated by increases in plasma concentrations of GPT and bilirubin, and hepatic triglyceride content (Hanzlik et al., 1978). It also produces acute histopathological changes in liver very similar to those induced by thioacetamide, i.e., depletion of glycogen in centrolobular hepatocytes as early as 6 hr after dosing progressing to
FIG. 8. Hammett plot of the plasma GPT response (units/ml) 24 hr after ip administration of 0.8 mmoYkg of various p-substituted thiobenzamide-S-oxides. Values are the mean ? SE for four rats. The slope of the correlation is -3.23.
severe centrolobular necrosis by 24 hr (Malvaldi, 1977; Chieli et al., 1979). From our initial work it appeared that the toxicity of thiobenzamide derivatives could be increased or decreased by para substituents which were electron-donating or -withdrawing, respectively. This was particularly intriguing because it suggested a link between the chemical properties of the thiobenzamides (or metabolites thereof) and the production of tissue injury. In particular the increase in toxicity by electron donating substituents would be understandable if oxidative biotransformation of thiobenzamide and its derivatives were required for production of toxicity in rive, as has been suggested for other thiocarbonyl compounds including thioacetamide (Hunter et al., 1977; Porter et al., 1979; Porter and Neal, 1978), carbon disulfide (De Matteis and Seawright, 1973; Dalvi et al., 1974; De Matteis, 1974), and various thiourea derivatives (De Matteis, 1974; Boyd and Neal, 1976). To investigate this aspect more thoroughly we have now determined the relative hepatotoxicity of five para-substituted thiobenzamides using increases in plasma GPT and bilirubin concentrations to assess liver injury. The results of this study appear
268
HANZLIK,
CASHMAN,
as Hammett plots in Figs. 1 and 2. Several features of these plots are particularly worthy of note. The excellent linear correlations between the toxic response and the Hammett sigma parameter, which characterizes the electron-donating or -withdrawing ability of the pnra-substituent (Lowry and Richardson, 1976), indicates that the relative toxicities of these compounds are indeed strongly dependent on their chemicat properties. Since no correlation was found between toxicity and the Hansch 7r parameter, which reflects the effects of the substituents on the lipid/water partitioning behavior of the molecules (Hansch et al.. 1973), it is most unlikely that the observed differences in toxicity result from differences in the absorption and/or distribution of the thiobenzamides. Rather, toxicity would appear to be linked to a chemical process such as oxidative biotransformation (vide i+.~). Furthermore. the fact that both groups of compounds at the two dosages used gave parallel Hammett plots (Figs. 1 and 2) strongly suggests that all five compounds share a common mechanism of action. The sign and magnitude of the slope of the Hammett correlation, i.e., the Hammett p value, also yield important information characterizing the chemical nature of the rate-limiting step in whatever sequence of events, as yet unknown. leads from the parent thioamide to the expression of toxicity in I~~~w. For the plasma GPT response the large negative p value of -3.42 suggests that some critical step leading to toxicity (1) involves the parent thiobenzamide or some closely relmted metabolite, and (2) involves an interaction which is very strongly facilitated by electron donation from ring substituents. In particular a large negative p value would be consistent with enzymatic S-oxidation as an obligatory step leading from thiobenzamide to the production of liver injury. Such p values have been observed in the chemical Soxidation of thiobenzophenones (Battaglia
AND
TRAIGER 2.0
FIG. (mg/lOO
1
9. Hammett plot ofthe plasma bilirubin response ml plasma)
24 hr after
ip administration
of
0.8 mmoVkg of various p-substituted thiobenzamideS-oxides. Values are the mean f SE for four rats. The slope of the line drawn is -0.2.
et al.,
1971). and thiobenzamide derivatives (J. R. Cashman, unpublished observations). Conversely, positive p values are observed for reactions involving nucleophilic attack on thiocarbonyl compounds (Drobnica and Augustin, 1965; Kardos et rd.. 1965), reflecting their facilitation by electron-withdrawing substituents. This point is especially significant in light of the importance usually ascribed to covalent binding of reactive metabolites (electrophiles) to celhilar macromolecules (nucleophiles) in cases of tissue injury due to chemicals (Jollow et ml., 1978). Many instances of tissue injury due to chemicals appear to result from oxidative biotransformation of the substance to a chemically reactive metabolite, often an electrophilic agent, which then alkylates various cellular nucleophiles leading uitimately to cell injury or death. In the present case the large negative rho values for the Hammett correlations in Figs. 1 and 2 should fzor be taken to imply that covalent binding of an electrophilic chemically reactive metabolite of thiobenzamide is not involved in the production of toxicity. They only imply that if such a covalent binding step is involved, it is not the rate-limiting step in the process leading from the thiobenzamide to the expression of toxicity. The failure of p-hydroxythiobenzamide to conform to the Hammett structure-
THIOBENZAMIDE DETOXICATION
BIOCHEMICAL
EVENTS
ALTERATIONS
DISTURBANCE
ASSESSMENT
AND RESPONSES;
OF HOMEOSTASIS
OF TOXICITY
FIG. 10. Hypothetical model relating the in viva disposition of TB and TBSO to the production of toxic responses. For details see discussion in text.
toxicity correlations presented in Figs. 1 and 2 can potentially be attributed to its ability to undergo rapid conjugation with glucuronic acid and/or sulfate, and thereby avoid Soxidative pathways leading to toxic metabolites, as mentioned above (Scheline et al., 1961). However, the lack of hepatotoxicity of 2,6-dichlorothiobenzamide is not likely to be due to a major shift in metabolism since its major metabolite, 2,6-dichlorobenzonitrile, most likely arises via S-oxidative metabolism. Nor can it be explained by the electron withdrawing effect of the two chlorines, since 2,6-dimethylthiobenzamide is equally nontoxic in the rat. The lack of toxicity of 2,6-disubstituted thiobenzamides can probably be attributed to (1) the ready decomposition of the S-oxides to the nitrile metabolite, which was previously shown not to be hepatotoxic in the rat (Griffiths et al., 1966), and/or (2) unfavorable steric effects of the substituents on a putative second S-oxidation step, as discussed
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further below. Further studies in progress should help to clarify this point. Both the electronic substituent effects discussed above and earlier results with inducers and inhibitors of drug metabolism suggested the possible involvement of enzymatic S-oxidation of thiobenzamide in the expression of its toxicity. Further support for this hypothesis comes from comparison of the time courses of plasma GPT and bilirubin and hepatic triglyceride concentrations following equimolar ip doses of TB and TBSO (Figs. 3, 4, and 5). The faster onset and/or greater intensity of these responses to TBSO is generally consistent with its obligatory involvement in liver injury by TB. However, before attempting to analyze TBSO vis-a-vis TB in greater detail, it is useful to consider the diagram in Fig. 10. Following ip administration of TB it must be absorbed and transported to the liver (ki). AS it is absorbed it can begin to undergo biotransformation. Thiobenzamide is known to be S-oxidized by a purified microsomal flavin-containing monooxygenase (Ziegler et al., 1979), and in vitro studies with rat liver microsomes in our laboratory have shown that TB is rapidly and extensively converted to TBSO (J. R. Cashman, unpublished observations), thus establishing the existence of the k, step in Fig. 10. Alternatively, of course, TBSO may be administered directly (kf ). Back-reduction of TBSO to TB (k-,) has not been specifically investigated but similar reductions have been observed in vivo with related thioamide-S-oxides (Johnston et al., 1967; Ammon et al., 1967; Porter and Neal, 1978). Further oxidation of TBSO to TBSO, (kz) is also a likely possibility (Hunter et al., 1977; Porter ef al., 1979). Potentially TB, TBSO, and TBSO, can each be “detoxified” (kdr kh, k$, respectively) by a variety of processes such as biotransformation or elimination. Presumably TB or its metabolites also interact in various deleterious ways with critical cellular targets
270
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CASHMAN,
such as enzymes or macromolecules, as represented by k,, k;, and k?. These latter events will perturb the normal homeostatic balance of the cell, and if the perturbation is large enough, it will be detected at some point by histological examination of the tissue and/or by measuring some biochemical parameter of cell function or integrity. Of course, it must be borne in mind that such attempts to assess toxicity are merely instantaneous glimpses of a complex dynamic process. In light of the above discussion one can now consider the “relative toxicities” of equimolar doses of TB and TBSO in terms of both the rate of onset and the ultimate intensity of tissue injury. If TBSO is an obligatory intermediate in toxic responses to TB, the actual response to its presence in the target cell will presumably depend on the balance between processes represented by k; and those represented by ki and k-,. Thus if some interaction of TBSO with the cell is very proximate to the measured index of toxicity one would certainly expect a more rapid response to TBSO than to TB. However, if the interaction of TBSO with the cell were many subsequent steps removed from the measured index of toxicity one might not detect much difference in onset of response between TBSO and TB, especially if the formation of TBSO from TB were faster than some of the subsequent steps leading to the measured index of toxicity (k, > ki). Finally if the processes represented by k; and k_, are relatively easily saturated, a rapid input of TBSO by direct administration is more likely to produce a greater response than a slower input from enzymatic oxidation of an equimolar amount of TB. The above model, while hypothetical, can help to rationalize numerous observations pertaining to hepatic injury by derivatives of TB and TBSO. For example, the faster onset and greater overall response of plasma bilirubin to TBSO, and the moderate but negative rho value for Fig. 2 are consistent
AND
TRAIGER
with an obligatory role for TBSO in interfering with biliary function leading to hyperbilirubinemia. The lack of substituent effects seen in Fig. 9 would logically result if TBSO derivatives did not require further oxidation in order to interfere with biliary function. The increase in plasma bilirubin due to TB has previously been shown to result from a decreased ability of the liver both to extract organic anions from plasma and to secrete them into bile (Vyas, 1979). Furthermore, the liver necrosis caused by TB does not per se result in hyperbilirubinemia, because doses of carbon tetrachloride producing even significantly greater increases in plasma GPT do not alter plasma bilirubin levels (Vyas, 1979). In contrast, it would appear that the plasma GPT response might require further oxidative metabolism of TBSO, perhaps to TBSO,. This could explain why the p values of Figs. 1 and 8 are essentially identical but much more negative than the p value for Fig. 2. i.e., if the second oxidation of TB, not the first, were rate limiting for the production of necrosis. Similar kinetic trends are seen in chemical oxidations of thioamides; the second oxidation step is always much slower than the first (Battaglia et al., 1971), and less reactive series of thioamides show much larger negative p values (i.e., greater dependence on electron donation from substituents) than the more reactive ones. That the GPT response to TBSO is slightly faster in onset but not significantly greater in overall intensity than that due to TB would also be consistent with an obligatory role for TBS02, since formation of the latter would be governed mainly by k, irrespective of whether TB or TBSO were administered. In some cases in which a toxic response is due to a metabolite of the parent drug, induction or inhibition of the required biotransformation leads to consistent and predictable alterations in the toxic responses. With agents such as bromobenzene, carbon tetrachloride, or acetaminophen such alterations in toxicity form a major part of the
THIOBENZAMIDE
evidence implicating a toxic metabolite (Jollow ef al., 1978). The toxic responses to TB and TBSO, however, are not altered in a consistent way by phenobarbital pretreatment, SKF-525A or NOI. One reason for this might simply be the greater complexity of events involved in the disposition of and biological response to TB and TBSO, e.g., the probable reversibility of the first oxidation of TB (which has no analogy in the simpler cases mentioned above), or the possible association of different toxic responses with different metabolites. Another important reason may be the involvement of the microsomal flavin-containing monooxygenase in the oxidation of TB (see above), and possibly of TBSO as well, because this enzyme does not respond to phenobarbital as do the cytochromes P-450, nor is it much inhibited by SKF-525A or NOI. Better understanding of the effects of inducers and inhibitors on the toxicity of TB and TBSO must await detailed investigation of their metabolism and disposition; such studies are presently under way in our laboratory. ACKNOWLEDGMENTS We are grateful to the National Institutes of Health (RR-5606) and the University of Kansas General Research Fund for their financial support of this work.
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DALVI, R. R., POORE, R. E., AND NEAL, R. A. (1974). Studies of the metabolism of carbon disulfide by rat liver microsomes. L$f Sci. 14, 1785- 17%. DE MATTEIS, F.. AND SEAWRIGHT, A. (1973). Oxidative metabolism of carbon disulphide by the rat. Effect of treatments which modify the liver toxicity of C&. Chem. -Biol. Interact. 7, 375-388. DE MATTEIS, F. (1974). Covalent binding of sulfur to microsomes and loss of cytochrome P-450 during the oxidative desulfurization of several chemicals. Mol. Phormacol. 10, 849-854. DROBNICA. L., AND AUGUSTIN, J. (l%S). Reactions of isothiocyanates with amino acids, peptides, and proteins. I. Kinetics of the reaction of aromatic isothiocyanates with glycine. Cot/. C:rch. ClrPtn. C0mm1tr2. 30, 99- 104. FAIRFUL. A. E. S., LOWE. J. L.. AND PEAK, D. A. ( 1952). A convenient reagent for the preparation of thioamides, and the thiohydrolysis of S-alkyl-isothiourea derivatives. J. Cltrm. Sot., 742-744. GRIFFITHS, M. H., Moss. J. A., ROSE, J. A., AND HATHWAY. D. E. (1966). The comparative metabolism of 2.6-dichlorothiobenzamide (Prefix) and 2,6-dichlorobenzonitrile in the dog and rat. Biochem. J. 98, 770-781. HANSCH. C., LEO. A., UNGER, S. H., KIM, K. H.. NIKAITANI. D., AND LIEN, E. J. (1973). Aromatic substituent constants for structure-activity correlations. J. Med. Chum. 16, 1207-1216. HANZLIK, R. P., VYAS. K. P., AND TRAIGER, G. J. (1978). Substituent effects on the hepatotoxicity of thiobenzamide derivatives in the rat. To.rico/. Appl. Phnrmacol. 46, 685-694. HUNTER, A. L.. HOLXHER, M. A., AND NEAL, R. A. ( 1977). Thioacetamide induced hepatic necrosis. I. Involvement of the mixed-function oxidase enzyme system. J. Pharmcrcol. Exp. Ther. 200, 439-448. JOHNSTON, J. P.. KANE, P. O., AND KIFGBY, M. R. (1967). The metabolism of ethionamide and its sulfoxide. .I. Phurm. Phnrmucol. 19, 1-9. JOLLOW, D. J., Kocsrs, J. J.. SNYDER, R.. AND VAINIO, H. (1978). Biological Reactive Intermedicttes. Plenum, New York. KAE~D~S, A. M., VOLKE, J., AND KRISTIAN. P. (1965). Polarography of phenyl isothiocyanates substituted in meta-position and kinetics of their reactions with hydroxyl ions. Co/l. Czech. Chem. Commun. 30, 931-938. LOWRY, R. H., AND RICHARDSON, K. S. (1976). Mechanism and Theory in Organic Chemistry, pp. 60-71. Harper & Row, New York. MALVALDI. G. (1977). Liver changes following thiobenzamide poisoning. Experientia 33, 1200-1201. PORTER, W. R., AND NEAL, R. A. (1978). Metabolism of thioacetamide and thioacetamide-S-oxide by rat liver microsomes. Drug Metub. Dispos. 6,379-388. PORTER, W. R., GUDZINOWECZ. M. J., AND NEAL,
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