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Bioactivation of carbon tetrachloride, chloroform and bromotrichloromethane: Role of cytochrome P-450
Pergamon Press
Life Sciences Vol . 20, pp . 1541-1548, 1977 . Printed in the U .S .A .
BIOACTIVATION OF CARBON TETRACHLORIDE, CHLOROFORM AND BROMOTRICHLOROMETHANE : ROLE OF CYTOCHROME P-450 I . Glenn Sipes*, Gopal Krishna and James R . Gillette Laboratory of Chemical Pharmacology, National Heart, Lung, And Blood Institute, National Institutes Of Health, Bethesda, MO .
20014
(Received in final form March 28, 1977) Summary In order to define the site of bioactivation of CC1 4 , CHC1 3 and CBrCl, in. the NADPH cytochrome c reductase-cytochrome P-450 coupled systems of liver microsomas, the 14 6-labeled hepatotoxins were incubated in vitro with isolated rat liver microsomas and a NADPH-generating system . TTie comment binding of radiolabel to microsomal protein was used as a measure of the conversion of the hepatotoxins to reactive intermediates . Omission of NADPH, incubation under CO :02 (8 :2) and addition of a cytochrome reductase specific When antisera mardedly reduced the covalent binding of all three compounds . cytochrome P-450 was reduced to less than 25% of normal by pretreatment of rats with allylisopropylacetamide (AIA), but cytochrome c reductase activity was unchanged, the covalent binding of CC1 4 , CHC1 2 , and CBrCI, was decreased by 63, 83, 70%, respectively . Incubation under an atmosphere of N2 enhanced the binding of CC1 4 , inhibited the binding of CHCI3 and did not influence the binding of CBrCI, . It is concluded that cytochrome P-450 is the site of bioactivation of these three compounds rather than NADPH cytochrome reductase and that CC1 4 bioactivation proceeds by cytochrome P-450 dependent reductive pathways, while CHC1 3 activation proceeds by cytochrome P-450 dependent oxidative pathways .
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The mechanism by which carbon tetrachloride (CC1 4 ) and related halomethanes produce hepatocellular necrosis has been the subject of numerous investigations . At present the prevailing theories state that these agents are first converted to reactive intermediates by enzymes present in the endoplasmic reticulum of the liver (1-3) . These active intermediates can then interact with liver macromolecules . This interaction results in an alteration of cellular integrity that ultimately leads to cell death (2,4) . For example, CC14 is converted to the trichloromethyl free radical ( " CC1,) (5) which covalently binds to lipid and protein (2,6,7) and also initiates peroxidation of polyenoic fatty acids (5,8) . Chloroform (CHC1 9 ) and bromotrichloromethane (CBrCl9) also bind covalently to liver macromolecules (9-11) and initiate lipid peroxidation as early events that precede morphological alterations (9,5,12) . Whether the covalent binding of reactive metabolites to tissue macromolecules causes toxic effects is debatable . Many investigators believe *Present address : Departments of Pharmacology, Toxicology, and Anesthesiology University of Arizona, Health Sciences Center, Tucson, Arizona 85724 1541
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that the trichloromethyl free radical formed from carbon tetrachloride and bromotrichloromethane initiates lipid peroxidation by reacting with polyenoic fatty acids to form chloroform and free radicals of the fatty acids . Thus in this mechanism lipid peroxidation would not result from the covalent binding to macromolecules . Even if this were the mechanism, however, the rate of covalent binding to macromolecules still can be used as an indirect measure of the concentration of the trichloromethyl free radical . The actual site in the endoplasmic reticulum where CC14, CHC13 and CBrCly are converted to highly reactive intermediates is not precisely defined . Slater and Sawyer (13) indicated that CC14 is converted to " CCl by the flavoprotein component of the NADPH-linked microsomal drug metabolizing system, commonly referred to as NADPH-cytochrome c reductase . However, several other investigators have suggested that the site of activation of CC1 4 is the cytochrome P-450/cytochrome P-450 reductase component of the microsomal system (14,15) . In order to define more adequately the site of activation of CC1 4 , CBrCl , and CH C13 we measured the in vitro covalent binding of these compounds to microsomal protein in the presencea NADPH cytochrome c reductase antisera or with microsomes from animals pretreated with allylisopropylacetamide (AIA) which specifically lowers the concentration of microsomal cytochrome P-450 . The results suggest that the cytochrome P-450 system is required for the conversion of CC14, CBrC13 and CHC13 to reactive intermediates and that NADPH supplies electrons to this system via cytochrome c reductase . Methods Male Sprague-Dawley rats (180-235 g) were pretreated once daily with phenobarbital (80 mg/kg, i .p .) for 4 days and then sacrificed by decapitation . Certain animals were injected with AIA (200 mg/kg s .c .) 75 min before sacrifice . Livers were removed and homogenized in cold 0 .02 M Tris-1 .15% KC1 buffer (pH 7 .4) and the microsomal fraction was obtained by differential centrifugation . The isolated microsomes were then resuspended in Tris-KC1 and recentrifuged at 100,000 x g for 1 hr . The microsomal pellet was suspended in Tris-KCl buffer and adjusted to the desired protein concentration as measured by the method of Gornall et al (16) . The amounts of cytochrome P-450 were determined as described by Omura and Sato (17) and the NADPH-cytochrome _c reductase activity was measured according to Phillips and Langdon (18) . The formation o active intermediates was studied by measuring the covalent binding of 4CC1 4 , 14 CHC1 and 14 CBrCl 3 to microsomal protein in an in vitro incubation system . The final incubation mixture (2 ml) consisted of râtTiver microsomes (0 .5 or 1 mg/ml protein), 0 .2mM NADP, 0 .124mM NADH, 2mM glucose-6-phosphate, 2mM nicotinamide, 2 units of glucose-6-phosphate dehydrogenase and 1mM of the 14C-substrates (specific activity 1 mCi/mmole) . The cytochrome c reductase antisera was included in certain experiments . A nonimmune sera was added to the corresponding controls . In both cases this represented the addition of 6.5 mg of protein to each vial . When incubations were performed under an atmosphere of N2 or CO :0 2 (8 :2) the vials were flushed for 5 min with the appropriate gas mixture and then stoppered with a thin rubber cap . The substrates were added with a Hamilton syringe through the cap and then immediately sealed . The mixtures were incubated with shaking in closed vials for 2 or 5 min at 37 ° C and then 1 ml of 10% TCA was added to stop the reaction and precipitate protein . The samples were then centrifuged and the resulting pellet resuspended in 5 ml of methanol :ether (3 :1) and heated to 60'C for 10 min and then centrifuged . This procedure was repeated 10 times with methanol :ether (3 :1) to remove any noncovently bound radioactivity . After aspiration of the final wash,the protein pellet was dissolved
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in 1 ml of 1N NaOH . Aliquots of the resulting solution were then removed for determination of covalently bound radioactivity and of protein by the method of Lowry (19) . Heat denatured microsomes were used to determine the extent of any nonenzymatically formed reactive intermediates . These samples contained less than 5 cpm above background and were subtracted from the appropriate sample . After pronase hydrolysis of the protein containing covalently bound 14 CC1 4 , 80% of the radioactivity was present in one unknown amino acid peak and the rest of the radioactivity in other peaks as determined by separation of amino acids by amino acid analysis . This indicated that the above washing procedure adequately removed all of the noncovalently bound radioactivity . The metabolism of ethylmorphine was determined in a 3 .0 ml incubation mixture consisting of microsomel protein (3 mg) and IOmM ethylmorphine, 5mM MgC1 2 and the above mentioned cofactors, enzyme and buffer . After incubation for 10 min at 37°C, the reaction was stopped with 1 .0 ml of ZnS04 (15%) and the amount of formaldehyde formed was measured according to the method of Nash (20) . All radioactive compounds were purchased from New England Nuclear Corp . The cytochrome c reductase antisera (immune serum) and the nonimmune serum were prepared and kindly supplied by Drs . H . Sasame and S . Thorgeirsson (21) . Allylisopropylacetamide was a gift of Dr . Wayne Levin of Hoffmann-La Roche, Inc . Student's t test was used to evaluate the significance of differences between the various groups . Results As shown in Table 1, the covalent binding of CBrCI 3 to protein in vitro was 15 times greater than that of CC1 4 and 24 times greater than CHCT3 , w en incubations were performed in air . It should be stressed that the microsomes used in these studies were obtained from phenobarbital pretreated animals, which enhances the binding of the substrates (9,11,22) . In an atmosphere of N 2 , however, the covalent binding of CC1 4 was increased 3-fold, while the binding of CHC1, was decreased by 66% . In these experiments the covalent binding of CBrCI 3 was unaffected by a N 2 atmosphere . But, in another study using microsomes from noninduced rats, incubation of CBrCI3 under an atmosphere of N 2 resulted in a 40% decrease in its binding to protein . Although we have performed numerous incubations to establish the effect of N 2 on the binding of CBrCI the data is variable and a definitive statement regarding its effect cannot be made . Incubation under an atmosphere of 80% CO (CO :0 2 -8 :2) markedly inhibited the covalent binding of CC1 4 and CHC1, . An atmosphere of 100% CO, was also effective in inhibiting the covalent binding of CM, and CHC1, . The covalent binding of CBrCI3 was much less affected by CO than the binding of CC1 4 and CHC1, ; an atmosphere of 80% CO decreased the covalent binding of CBrCl, by 40% and an atmosphere of 100% decreased it by 38% . SKF 525-A inhibits the biotransformation and covalent binding of many drugs and other xenobiotics (23) . In these experiments addition of SKF 525-A to the incubation media significatnly decreased the covalent binding of CHC1,, was ineffective agains the covalent binding of CC1 4 and increased (40%) the covalent binding of CBrCl, (Table 1) .
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Table 1 Effects of NZ or CO :02 Atmosphere, Omission of NADPH and Addition Of Glutathione on the in vitro Covalent Binding of CC1 4 CHC1, and CBrC1 3 to Rat Liver Microsomal Protein Incubati on Conditions
Substrates CC1 4
Atmosphere Air N2 CO :02(8 :2) CO 100% SKF 525A (0 .5mM)
CHC1 3
CBrCl,
picomoles 14 C-bound/mg protein microsomal per min 97 310 18 40
± 10 ± 51* ± 1* t 6*
109 ± 5
59 21 20 11
± 5 ± 1* f 1* ± 1*
7 ± 1*
1456 1370 853 560
± 66 ± 143 t 62** ± 98**
2105 ± 159**
Glutathione (3mM)
17 t 2*
15 t 2*
218 ± 25*
Omission of NADPH
6 ± 1*
3 ± 0*
65 ± 13*
Incubations were performed at 37°C using 14C_ labeled substrates at final concentrations of 1 x 10 -3 M (specific activity lmC/mmole) as described in Methods . Microsomes from phenobarbital pretreated rats were resent at a final protein concentration of lmg/ml . ach value is the mean ± S .E .M . for 4 experiments . *p < .01 ; ** p < .05 as compared to incubation in air . Addition of glutathione to the incubation medium resulted in approximately an 80% inhibition of binding for all three compounds . Similarly, the omission of the NADPH generating system from the incubations nearly abolished the binding of CC1 4 , CHCl ., and CBrCl 3 (Table 1) . Highly specific antisera against microsomal cytochrome c reductase markedly inhibited the covalent binding of all three hepatotoxins to liver microsomal protein (Table 2) . This inhibition was observed when incubations were performed under air or N2 . We did not test the effect of the antisera against CHC1 3 binding under N2 . since there was very little binding of CHC1, under anaerobic conditions, The-magnitude of the decrease in the covalent binding appeared to be similar to the inhibition of cytochrome c reductase and to the decrease in ethylmorphine N-demethylase activity, which is a NADPHdependent cytochrome P-450 coupled enzyme system (Table 2) . As indicated in Table 3, pretreatment of rats with allylisopropylacetamide greatly reduced the level of hepatic microsomal cytochrome P-450 . This reduction in cytochrome P-450 content is reflected in a similar decrease in the activity of ethylmorphine N-demethylase . However, NADPH-cytochrome c reductase, was unaffected by AIA pretreatment . The pretreatment with AIT also reduced the in vitro covalent binding of CC1 4 , CBrCl, and CHCl, to liver microsomal protein . The degree in the reduction of the covalent binding was of the same magnitude as the reduction in cytochrome P-450 content .
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Table 2 Effects of Cytochrome c Reductase Antibody on the in vitro Covalent Binding of CC1 4 , CHC1, and CBrCI, to Protein and on MicrosomeTÉtFylmorphine-NDemethylase and Cytochrome c Reductase Activities Substrates
Nonimmune Serum
Antibody Serum
picomoles 1 4C-bou nd per mg total protein per min
Decrease
CCl A r Atmosphere N2 Atmosphere
22 ± 1 80 ± 3
5 ± 1* 26 ± 5*
77 67
CHC1 r Atmosphere
11 ± 0
3 t 1*
73
CBrCl Atmosphere NZ Atmosphere
181 t 7 126 ± 13
60 ± 4* 87 ± 10*
66 51
N-demethylation of 27 .8 ± 2 Ethylmorphine nanomoles HCHO "mg -1 " min - '
6 .8 t 1*
75
Cytochrome 137 ± 17 reductase activity nanomoles-mg - ' " min -1
32 .8 ± 4*
76
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Incubations contained 0.5mg/ml microsomal protein plus 6 .25mg serum protein for substrates CC14, CHC1, and CBrCI, and lmg/ml microsomal protein plus 12 .5mg serum proteins for ethylmorphine . Microsomes were obtained from phenobarbital pretreated rats . Binding is expressed per mg of total protein since microsomal protein was not separated from the added serum protein . Each value is the mean ± S .E .M . for 4 experiments . *p < .O1 as compared to mean for nonimmune serum. Discussion . CC14, CHC1, and CSrCl, are known to produce liver necrosis, but the actual mechanism by which necrosis is produced is not completely resolved . However, it is generally agreed that the initial event in the production of tissue damage is the bioactivation of these chemicals into highly reactive intermediates (5) . For example, CC14 and CBrCl, are thought to be converted to the trichloromethyl ( " CC1,) free radical (11,22) . These free radicals can then attack various tissue components which result in the initiation of peroxidation of polyunsaturated fatty acids (3,8) and the covalent binding of the reactive intermediates to protein and lipids (2,6) . In the studies reported here it was shown that the three hepatotoxins covalently bind to microsomal protein . Of major importance is the high degree of covalent binding produced by CBrCl, . This is in agreement with the observations that CBrCl, is 30 to 40 times more hepatotoxic than CC14 (24,26) and its ability to induce lipoperoxidation is 200 times greater than that of CC1 4 (12) . The dramatic increase in the covalent binding may result from the lower bond dissociation energy of the C-Br bond as compared to the C-C1
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Table 3 Effects of Allylisopropylacetamide (AIA) Pretreatment on the in vitro Covalent Binding of CC1 4 , CBrCl 3 and CHC1 3 to Microsomal Prote Substrate
Saline Pretreatment
AIA Pretreatment
% Decrease
picomoles/mg microsomal protein per min 167 CC1 4 ±
8
61
± 8*
63
2959 CBrC1 3 ± 131
886
±13*
70
22
± 1*
83
130 CHC1 3 ±
17
N-demethylation of Ethylmorphine nanomoles HCHOmgri-min-1 Levels of Cytochrome P-450 nanomoles*mg-1
2 .23 t
Cytochrome c 104 .1 Reductase Activity nanomoles " mg-1 "min-1
0.18
0 .52* ± 0 .02
76.
9.2
99 .1 t 2.1
5
Each value is the mean t S .E .M . for 4 experiments . *p q .O1 as compared to saline pretreatment . AIA (200mg/kg, subcut . i n saline) was administered to phenobarbital induced rats 75 minutes before isolation of liver microsomal enzymes . Microsomal protein concentration was 1 mg/ml . Incubation conditions are as described in Methods . bond (5) . This would favor the more rapid formation of the trichloromethyl free radical from CBrCl, . In addition, Bini et al (27) have suggested that another free radical (-CBrCl2) is formed from-MrZ"I 3 which may be involved in its induction of hepatotoxicity . The role of cytochrome P-450 in the bioactivation of these hepatotoxins is substantiated by the data presented in Tables 1, 2 and 3 of this report . The reduction in covalent binding by an atmosphere containing-80% 00 :20% 02 suggests a cytochrome P-450 dependent pathway . Similarly, the 95% reduction in binding observed when NADPH was omitted from the incubation medium is indicative of a NADPH cytochrome c reductase-cytochrome P-450 coupled reaction . This is further substantiated by the reduction in binding when cytochrome c reductase antisera was added to the microsomal incubations . The inhibitory effects of the NADPH cytochrome c reductase antisera could be interpreted as supporting the conclusion by Slater and Sawyer (13) that the point of interaction between CC14 and the mixed function oxygenase system for the production of "CC1, is at the NADPH cytochrome c reductase flavoprotein . However, the covalent binding of CC1 4 , CHC1 3 ana CBrCl, was reduced by 63% or more when microsomal cytochrome P-450 was decreased by 76% by pretreatment of rats with AIA, even though this treatment did not change the activity of cytochrome c reductase . These data thus suggest that cytochrome P-450 is the site of conversion of CC1 4 , CHC12 and CBrCI, to reactive intermediates .
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Observations by other investigators also strongly sup ort the role of cytochrome P-450 in the bioactivation of CC14 . Glende (28~ concluded that cytochrome P-450 was instrumental in mediating the CC1 4 -induced .'destruction of cytochrome P-450 and related microsomal enzymatic activities . He was able to separate the roles of cytochrome P-450 and NADPH cytochrome c reductase by pretreating animals with small doses of CC1 4 . This treatment which decreased cytochrome P-450 levels by 80%, but did not change the NADPH cytochrome c reductase activity, protected rats from the toxic effects of a lethal dose of CC14 . Carlson (29) correlated a protection of CC1 4 induced hepatotoxicity in rats pretreated with methylmercury hydroxide with the methylmercury induced reduction in hepatic microsomal cytochrome P-450 . Similarly, Suarez and Bhonsle (30) observed a protective effect by cobaltous chloride pretreatment against CC1 4 -induced lipoperoxidation and increases in serum enzymes . They attributed this protection to a cobaltous chloride induced decrease of cytochrome P-450 . The effects of the various atmospheres on the covalent binding of these compounds (Table 1) suggest that CHC1 3 is either metabolized differently than CC1 4 and CBrCl, or else the steady state levels of their reactive intermediates are affected differently by N2 or 02 . Regarding the latter point, the reactive intermediate(s) from CC1 4 may be inactivated by an 02 dependent process and those of CHC13 by a reductive process that is not OZ dependent . An atmosphere favoring increased decomposition of the reactive intermediates to inactive species would be observed as a reduction in binding . However, we favor the alternative proposal that the reduction in binding reflects different pathways of bioactivation . The reduction in binding of CHC1, by an atmosphere of N2 suggests that its bioactivation is mediated by a cytochrome P-450 oxidative pathway and that oxygen is probably incorporated into the reactive intermediate . This conclusion is further supported by the studies of Brown et al (31) who reported that in mice 80% of administered 14 CHC1, was recovered as 14 C02 " Although the identity of the intermediate is unknown, it could be trichloromethanol, which would decompose to phosgene (COC1 2 ) . Additional support for the classical cytochrome P-450 oxidative pathway in CHC1, bioactivation are the inhibitory effect of SKF 525-A on its binding (Table 1) and the seven fold increase in binding when liver microsomas are isolated from phenobarbital treated rats (9) . It is noteworthy that an atmosphere of N2 effects the bioactivation and/ or binding of CC1 4 , CHC1 3 and CBrCl, differently . Uehleke et al (32) reported that anaerobic conditions greatly enhanced the binding of CT4to liver endoplasmic reticulum proteins, as was observed in these studies . The enhanced binding of CC1 4 under N 2 suggests that CC1 4 is metabolized by cytochrome P-450 mediated reductive pathways and-that 02 inhibits biotransformation by competing for reducing equivalents . In many reductive pathways, SKF 525-A is known to be an ineffective inhibitor of drug metabolism and may actually increase the rate of reduction (23) possibly by increasing the rate of comsumption of trace amounts of oxygen remaining in the incubation mixtures . In this study SKF 525-A enhanced the binding of CBrCl, by 44% and was ineffective against CC1 4 . It was not possible to demonstrate an increase in binding of CBrCl, under N2, but all other aspects of its bioactivation and binding are similar to CC1 4 . The role of reduced glutathione in bioactivation and covalent binding of these three hepatotoxins remains unknown . It is extremely effective in decreasing the covalent binding . In phenobarbital treated animals CHC1,
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depletes liver glutathione presumably by the formation of a conjugate between the reactive intermediate (COC1 2 ) and glutathione (9,10) . However, it has not been possible to detect a decrease in the liver glutathione levels following administration of CC1 4 or CBrCl,(10) . Nevertheless, it seems possible that glutathione may decrease the levels of " CCl, by reducing it to chloroform and that the oxidized glutathione may be reduced back to glutahione by glutathione reductase . Whether this mechanism occurs in living animals, however, remains to be elucidated . References 1. 2. 3. 4. 5. 6. 7. 8. 9. 10 . 11 . 12 . 13 . 14 . 15 . 16 . 17 . 18 . 19 . 20 . 21 . 22 . 23 . 24 . 25 . 26 . 27 . 28 . 29 . 30 . 31 . 32 .
T . Slater, Nature 209 36 (1966) . E .S . Reynolds, J . Rlarmacol . Exp . Therap . 155 117-126 (1967) . R .O . Recknagel, Pharmacol . Rev . 19 145-2080-967) . R .O . Recknagel .an d A .K . Ghoshal,Tab . Invest . 15 132-148 (1966) . T .F . Slater, Free Radical Mechanisms in Tissuernjury, Pion Limited, London (1972) . K .S . Rao and R .O . Recknagel, Expt . Mol . Path . 10 219-228 (1969) . J .A . Castro and M .I . Diaz Gomez, Toxicol . Appl .Pharmacol . 23 541-552 (1972) . R .O . Recknagel and A .K . Ghoshal, Exp . Mol . Path . 5 413-426 (1966) . B .R . Brown, I .G . Sipes and A .M . Sagalyn, Anesthesiology 41 554-561 (1974) . E .L . Docks, and G . Krishna, Exp . Mol . Path . 24 13-22 (97T) . G . Krishna, I .G . Sipes and J .R . Gillette, Pharmacologist 15 260 (1973) . R .R . Koch, E .A . Glende Jr ., and R .O . Recknagel, Biochem . Phiarmacol . 23 2907-2915 (1974) . T .F . Slater and B .C . Sawyer, Biochem . J . 123 815-821 (1971) . N . D'Acosta, J .A . Castra, M .I . Diaz Gomez,¬ .C . de Ferregra, C .R . Castro and O .M . de Fenos, Res . Commun . Chem . Path . Pharmacol . 6 175-183 (1973) . 0 . Reiner, and H . Wehleke, Hoppe-Seylers Z . Physiol . Chem . 352 1048-1052 (1971) . A .G . Gornall, C .J . Bardawell and M .M . David, J . Biol . Chem . 177 751-766 (1946) . T . Omura and R . Sato, J . BIol . Chem . 239 2370-2378 (1964) . A . Phillips and R . Langdon, J . .Biol . Chem . 237 2652-2660 (1962) . 0 . Lowry, N . Rosebrough, A . Farr, R . Randall,J . Biol . Chem . 193 265-275 (1951) . I . Nash, Biochem . J . 55 416-421 (1953) . S .S . Thorgeirsson, D .J. Jollow, H .A . Sasame, I . Green and J .R . Mitchell, Mol . Pharmacol . 9 398-404 (1973) . K .f. . Ilett, W .D . Reid, I .G . Sipes and G . Krishna, Exp . Mol . Path . 19 215-229 (1973) . J .R . Gillette, Reductive Enzymes in Concepts in Biochemical Pharmacology (PP 350-361) Eds . B .B . Brodie and J .R . Gillette, Springer-Verlag, New York (1971) . E . Burdino, E . Gravela, G . Ugazio, V . Vannini, and A . Calligaro, Agents and Actions 4 244-253 (1973) . J .R . Gillette, Biochem . Pharmacol . 23 2785-2794 (1974) . I .G . Sipes and G . Krishna--manuscript in preparation . A . Bini, G . Vecchi, G . Vivoli, V . Vannini and C . Cessi, Pharmacol . Res . Comm . 7 143-149 (1975) . E .A . GTende, Jr ., Biochem . Pharmacol . 21 169761702 (1972) . G .P . Carlson, Toxicology 183-89 (1975] K .A . Suarez and P . Bhonsle, Toxicol . Appl . Pharmacol . 37 23-27 (1976) . D .M . Brown, P .F . Langley, D . Smith and D .C . Taylor, Xenobiotica 4 151-163 (1974) . H . Uehleke, K .H . Hellmer and S . Tabarelli, Xenobiotica 3 1-11 (1973) .
Life Sciences Vol . 20, pp . 1541-1548, 1977 . Printed in the U .S .A .
BIOACTIVATION OF CARBON TETRACHLORIDE, CHLOROFORM AND BROMOTRICHLOROMETHANE : ROLE OF CYTOCHROME P-450 I . Glenn Sipes*, Gopal Krishna and James R . Gillette Laboratory of Chemical Pharmacology, National Heart, Lung, And Blood Institute, National Institutes Of Health, Bethesda, MO .
20014
(Received in final form March 28, 1977) Summary In order to define the site of bioactivation of CC1 4 , CHC1 3 and CBrCl, in. the NADPH cytochrome c reductase-cytochrome P-450 coupled systems of liver microsomas, the 14 6-labeled hepatotoxins were incubated in vitro with isolated rat liver microsomas and a NADPH-generating system . TTie comment binding of radiolabel to microsomal protein was used as a measure of the conversion of the hepatotoxins to reactive intermediates . Omission of NADPH, incubation under CO :02 (8 :2) and addition of a cytochrome reductase specific When antisera mardedly reduced the covalent binding of all three compounds . cytochrome P-450 was reduced to less than 25% of normal by pretreatment of rats with allylisopropylacetamide (AIA), but cytochrome c reductase activity was unchanged, the covalent binding of CC1 4 , CHC1 2 , and CBrCI, was decreased by 63, 83, 70%, respectively . Incubation under an atmosphere of N2 enhanced the binding of CC1 4 , inhibited the binding of CHCI3 and did not influence the binding of CBrCI, . It is concluded that cytochrome P-450 is the site of bioactivation of these three compounds rather than NADPH cytochrome reductase and that CC1 4 bioactivation proceeds by cytochrome P-450 dependent reductive pathways, while CHC1 3 activation proceeds by cytochrome P-450 dependent oxidative pathways .
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The mechanism by which carbon tetrachloride (CC1 4 ) and related halomethanes produce hepatocellular necrosis has been the subject of numerous investigations . At present the prevailing theories state that these agents are first converted to reactive intermediates by enzymes present in the endoplasmic reticulum of the liver (1-3) . These active intermediates can then interact with liver macromolecules . This interaction results in an alteration of cellular integrity that ultimately leads to cell death (2,4) . For example, CC14 is converted to the trichloromethyl free radical ( " CC1,) (5) which covalently binds to lipid and protein (2,6,7) and also initiates peroxidation of polyenoic fatty acids (5,8) . Chloroform (CHC1 9 ) and bromotrichloromethane (CBrCl9) also bind covalently to liver macromolecules (9-11) and initiate lipid peroxidation as early events that precede morphological alterations (9,5,12) . Whether the covalent binding of reactive metabolites to tissue macromolecules causes toxic effects is debatable . Many investigators believe *Present address : Departments of Pharmacology, Toxicology, and Anesthesiology University of Arizona, Health Sciences Center, Tucson, Arizona 85724 1541
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that the trichloromethyl free radical formed from carbon tetrachloride and bromotrichloromethane initiates lipid peroxidation by reacting with polyenoic fatty acids to form chloroform and free radicals of the fatty acids . Thus in this mechanism lipid peroxidation would not result from the covalent binding to macromolecules . Even if this were the mechanism, however, the rate of covalent binding to macromolecules still can be used as an indirect measure of the concentration of the trichloromethyl free radical . The actual site in the endoplasmic reticulum where CC14, CHC13 and CBrCly are converted to highly reactive intermediates is not precisely defined . Slater and Sawyer (13) indicated that CC14 is converted to " CCl by the flavoprotein component of the NADPH-linked microsomal drug metabolizing system, commonly referred to as NADPH-cytochrome c reductase . However, several other investigators have suggested that the site of activation of CC1 4 is the cytochrome P-450/cytochrome P-450 reductase component of the microsomal system (14,15) . In order to define more adequately the site of activation of CC1 4 , CBrCl , and CH C13 we measured the in vitro covalent binding of these compounds to microsomal protein in the presencea NADPH cytochrome c reductase antisera or with microsomes from animals pretreated with allylisopropylacetamide (AIA) which specifically lowers the concentration of microsomal cytochrome P-450 . The results suggest that the cytochrome P-450 system is required for the conversion of CC14, CBrC13 and CHC13 to reactive intermediates and that NADPH supplies electrons to this system via cytochrome c reductase . Methods Male Sprague-Dawley rats (180-235 g) were pretreated once daily with phenobarbital (80 mg/kg, i .p .) for 4 days and then sacrificed by decapitation . Certain animals were injected with AIA (200 mg/kg s .c .) 75 min before sacrifice . Livers were removed and homogenized in cold 0 .02 M Tris-1 .15% KC1 buffer (pH 7 .4) and the microsomal fraction was obtained by differential centrifugation . The isolated microsomes were then resuspended in Tris-KC1 and recentrifuged at 100,000 x g for 1 hr . The microsomal pellet was suspended in Tris-KCl buffer and adjusted to the desired protein concentration as measured by the method of Gornall et al (16) . The amounts of cytochrome P-450 were determined as described by Omura and Sato (17) and the NADPH-cytochrome _c reductase activity was measured according to Phillips and Langdon (18) . The formation o active intermediates was studied by measuring the covalent binding of 4CC1 4 , 14 CHC1 and 14 CBrCl 3 to microsomal protein in an in vitro incubation system . The final incubation mixture (2 ml) consisted of râtTiver microsomes (0 .5 or 1 mg/ml protein), 0 .2mM NADP, 0 .124mM NADH, 2mM glucose-6-phosphate, 2mM nicotinamide, 2 units of glucose-6-phosphate dehydrogenase and 1mM of the 14C-substrates (specific activity 1 mCi/mmole) . The cytochrome c reductase antisera was included in certain experiments . A nonimmune sera was added to the corresponding controls . In both cases this represented the addition of 6.5 mg of protein to each vial . When incubations were performed under an atmosphere of N2 or CO :0 2 (8 :2) the vials were flushed for 5 min with the appropriate gas mixture and then stoppered with a thin rubber cap . The substrates were added with a Hamilton syringe through the cap and then immediately sealed . The mixtures were incubated with shaking in closed vials for 2 or 5 min at 37 ° C and then 1 ml of 10% TCA was added to stop the reaction and precipitate protein . The samples were then centrifuged and the resulting pellet resuspended in 5 ml of methanol :ether (3 :1) and heated to 60'C for 10 min and then centrifuged . This procedure was repeated 10 times with methanol :ether (3 :1) to remove any noncovently bound radioactivity . After aspiration of the final wash,the protein pellet was dissolved
Vol . 20, No . 9, 1977
Bioactivation of Hapatotoxins
154 3
in 1 ml of 1N NaOH . Aliquots of the resulting solution were then removed for determination of covalently bound radioactivity and of protein by the method of Lowry (19) . Heat denatured microsomes were used to determine the extent of any nonenzymatically formed reactive intermediates . These samples contained less than 5 cpm above background and were subtracted from the appropriate sample . After pronase hydrolysis of the protein containing covalently bound 14 CC1 4 , 80% of the radioactivity was present in one unknown amino acid peak and the rest of the radioactivity in other peaks as determined by separation of amino acids by amino acid analysis . This indicated that the above washing procedure adequately removed all of the noncovalently bound radioactivity . The metabolism of ethylmorphine was determined in a 3 .0 ml incubation mixture consisting of microsomel protein (3 mg) and IOmM ethylmorphine, 5mM MgC1 2 and the above mentioned cofactors, enzyme and buffer . After incubation for 10 min at 37°C, the reaction was stopped with 1 .0 ml of ZnS04 (15%) and the amount of formaldehyde formed was measured according to the method of Nash (20) . All radioactive compounds were purchased from New England Nuclear Corp . The cytochrome c reductase antisera (immune serum) and the nonimmune serum were prepared and kindly supplied by Drs . H . Sasame and S . Thorgeirsson (21) . Allylisopropylacetamide was a gift of Dr . Wayne Levin of Hoffmann-La Roche, Inc . Student's t test was used to evaluate the significance of differences between the various groups . Results As shown in Table 1, the covalent binding of CBrCI 3 to protein in vitro was 15 times greater than that of CC1 4 and 24 times greater than CHCT3 , w en incubations were performed in air . It should be stressed that the microsomes used in these studies were obtained from phenobarbital pretreated animals, which enhances the binding of the substrates (9,11,22) . In an atmosphere of N 2 , however, the covalent binding of CC1 4 was increased 3-fold, while the binding of CHC1, was decreased by 66% . In these experiments the covalent binding of CBrCI 3 was unaffected by a N 2 atmosphere . But, in another study using microsomes from noninduced rats, incubation of CBrCI3 under an atmosphere of N 2 resulted in a 40% decrease in its binding to protein . Although we have performed numerous incubations to establish the effect of N 2 on the binding of CBrCI the data is variable and a definitive statement regarding its effect cannot be made . Incubation under an atmosphere of 80% CO (CO :0 2 -8 :2) markedly inhibited the covalent binding of CC1 4 and CHC1, . An atmosphere of 100% CO, was also effective in inhibiting the covalent binding of CM, and CHC1, . The covalent binding of CBrCI3 was much less affected by CO than the binding of CC1 4 and CHC1, ; an atmosphere of 80% CO decreased the covalent binding of CBrCl, by 40% and an atmosphere of 100% decreased it by 38% . SKF 525-A inhibits the biotransformation and covalent binding of many drugs and other xenobiotics (23) . In these experiments addition of SKF 525-A to the incubation media significatnly decreased the covalent binding of CHC1,, was ineffective agains the covalent binding of CC1 4 and increased (40%) the covalent binding of CBrCl, (Table 1) .
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Table 1 Effects of NZ or CO :02 Atmosphere, Omission of NADPH and Addition Of Glutathione on the in vitro Covalent Binding of CC1 4 CHC1, and CBrC1 3 to Rat Liver Microsomal Protein Incubati on Conditions
Substrates CC1 4
Atmosphere Air N2 CO :02(8 :2) CO 100% SKF 525A (0 .5mM)
CHC1 3
CBrCl,
picomoles 14 C-bound/mg protein microsomal per min 97 310 18 40
± 10 ± 51* ± 1* t 6*
109 ± 5
59 21 20 11
± 5 ± 1* f 1* ± 1*
7 ± 1*
1456 1370 853 560
± 66 ± 143 t 62** ± 98**
2105 ± 159**
Glutathione (3mM)
17 t 2*
15 t 2*
218 ± 25*
Omission of NADPH
6 ± 1*
3 ± 0*
65 ± 13*
Incubations were performed at 37°C using 14C_ labeled substrates at final concentrations of 1 x 10 -3 M (specific activity lmC/mmole) as described in Methods . Microsomes from phenobarbital pretreated rats were resent at a final protein concentration of lmg/ml . ach value is the mean ± S .E .M . for 4 experiments . *p < .01 ; ** p < .05 as compared to incubation in air . Addition of glutathione to the incubation medium resulted in approximately an 80% inhibition of binding for all three compounds . Similarly, the omission of the NADPH generating system from the incubations nearly abolished the binding of CC1 4 , CHCl ., and CBrCl 3 (Table 1) . Highly specific antisera against microsomal cytochrome c reductase markedly inhibited the covalent binding of all three hepatotoxins to liver microsomal protein (Table 2) . This inhibition was observed when incubations were performed under air or N2 . We did not test the effect of the antisera against CHC1 3 binding under N2 . since there was very little binding of CHC1, under anaerobic conditions, The-magnitude of the decrease in the covalent binding appeared to be similar to the inhibition of cytochrome c reductase and to the decrease in ethylmorphine N-demethylase activity, which is a NADPHdependent cytochrome P-450 coupled enzyme system (Table 2) . As indicated in Table 3, pretreatment of rats with allylisopropylacetamide greatly reduced the level of hepatic microsomal cytochrome P-450 . This reduction in cytochrome P-450 content is reflected in a similar decrease in the activity of ethylmorphine N-demethylase . However, NADPH-cytochrome c reductase, was unaffected by AIA pretreatment . The pretreatment with AIT also reduced the in vitro covalent binding of CC1 4 , CBrCl, and CHCl, to liver microsomal protein . The degree in the reduction of the covalent binding was of the same magnitude as the reduction in cytochrome P-450 content .
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Vol. 20, No . 9, 1977
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Table 2 Effects of Cytochrome c Reductase Antibody on the in vitro Covalent Binding of CC1 4 , CHC1, and CBrCI, to Protein and on MicrosomeTÉtFylmorphine-NDemethylase and Cytochrome c Reductase Activities Substrates
Nonimmune Serum
Antibody Serum
picomoles 1 4C-bou nd per mg total protein per min
Decrease
CCl A r Atmosphere N2 Atmosphere
22 ± 1 80 ± 3
5 ± 1* 26 ± 5*
77 67
CHC1 r Atmosphere
11 ± 0
3 t 1*
73
CBrCl Atmosphere NZ Atmosphere
181 t 7 126 ± 13
60 ± 4* 87 ± 10*
66 51
N-demethylation of 27 .8 ± 2 Ethylmorphine nanomoles HCHO "mg -1 " min - '
6 .8 t 1*
75
Cytochrome 137 ± 17 reductase activity nanomoles-mg - ' " min -1
32 .8 ± 4*
76
c
Incubations contained 0.5mg/ml microsomal protein plus 6 .25mg serum protein for substrates CC14, CHC1, and CBrCI, and lmg/ml microsomal protein plus 12 .5mg serum proteins for ethylmorphine . Microsomes were obtained from phenobarbital pretreated rats . Binding is expressed per mg of total protein since microsomal protein was not separated from the added serum protein . Each value is the mean ± S .E .M . for 4 experiments . *p < .O1 as compared to mean for nonimmune serum. Discussion . CC14, CHC1, and CSrCl, are known to produce liver necrosis, but the actual mechanism by which necrosis is produced is not completely resolved . However, it is generally agreed that the initial event in the production of tissue damage is the bioactivation of these chemicals into highly reactive intermediates (5) . For example, CC14 and CBrCl, are thought to be converted to the trichloromethyl ( " CC1,) free radical (11,22) . These free radicals can then attack various tissue components which result in the initiation of peroxidation of polyunsaturated fatty acids (3,8) and the covalent binding of the reactive intermediates to protein and lipids (2,6) . In the studies reported here it was shown that the three hepatotoxins covalently bind to microsomal protein . Of major importance is the high degree of covalent binding produced by CBrCl, . This is in agreement with the observations that CBrCl, is 30 to 40 times more hepatotoxic than CC14 (24,26) and its ability to induce lipoperoxidation is 200 times greater than that of CC1 4 (12) . The dramatic increase in the covalent binding may result from the lower bond dissociation energy of the C-Br bond as compared to the C-C1
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Table 3 Effects of Allylisopropylacetamide (AIA) Pretreatment on the in vitro Covalent Binding of CC1 4 , CBrCl 3 and CHC1 3 to Microsomal Prote Substrate
Saline Pretreatment
AIA Pretreatment
% Decrease
picomoles/mg microsomal protein per min 167 CC1 4 ±
8
61
± 8*
63
2959 CBrC1 3 ± 131
886
±13*
70
22
± 1*
83
130 CHC1 3 ±
17
N-demethylation of Ethylmorphine nanomoles HCHOmgri-min-1 Levels of Cytochrome P-450 nanomoles*mg-1
2 .23 t
Cytochrome c 104 .1 Reductase Activity nanomoles " mg-1 "min-1
0.18
0 .52* ± 0 .02
76.
9.2
99 .1 t 2.1
5
Each value is the mean t S .E .M . for 4 experiments . *p q .O1 as compared to saline pretreatment . AIA (200mg/kg, subcut . i n saline) was administered to phenobarbital induced rats 75 minutes before isolation of liver microsomal enzymes . Microsomal protein concentration was 1 mg/ml . Incubation conditions are as described in Methods . bond (5) . This would favor the more rapid formation of the trichloromethyl free radical from CBrCl, . In addition, Bini et al (27) have suggested that another free radical (-CBrCl2) is formed from-MrZ"I 3 which may be involved in its induction of hepatotoxicity . The role of cytochrome P-450 in the bioactivation of these hepatotoxins is substantiated by the data presented in Tables 1, 2 and 3 of this report . The reduction in covalent binding by an atmosphere containing-80% 00 :20% 02 suggests a cytochrome P-450 dependent pathway . Similarly, the 95% reduction in binding observed when NADPH was omitted from the incubation medium is indicative of a NADPH cytochrome c reductase-cytochrome P-450 coupled reaction . This is further substantiated by the reduction in binding when cytochrome c reductase antisera was added to the microsomal incubations . The inhibitory effects of the NADPH cytochrome c reductase antisera could be interpreted as supporting the conclusion by Slater and Sawyer (13) that the point of interaction between CC14 and the mixed function oxygenase system for the production of "CC1, is at the NADPH cytochrome c reductase flavoprotein . However, the covalent binding of CC1 4 , CHC1 3 ana CBrCl, was reduced by 63% or more when microsomal cytochrome P-450 was decreased by 76% by pretreatment of rats with AIA, even though this treatment did not change the activity of cytochrome c reductase . These data thus suggest that cytochrome P-450 is the site of conversion of CC1 4 , CHC12 and CBrCI, to reactive intermediates .
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Bioactivation of Hapatotoxins
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Observations by other investigators also strongly sup ort the role of cytochrome P-450 in the bioactivation of CC14 . Glende (28~ concluded that cytochrome P-450 was instrumental in mediating the CC1 4 -induced .'destruction of cytochrome P-450 and related microsomal enzymatic activities . He was able to separate the roles of cytochrome P-450 and NADPH cytochrome c reductase by pretreating animals with small doses of CC1 4 . This treatment which decreased cytochrome P-450 levels by 80%, but did not change the NADPH cytochrome c reductase activity, protected rats from the toxic effects of a lethal dose of CC14 . Carlson (29) correlated a protection of CC1 4 induced hepatotoxicity in rats pretreated with methylmercury hydroxide with the methylmercury induced reduction in hepatic microsomal cytochrome P-450 . Similarly, Suarez and Bhonsle (30) observed a protective effect by cobaltous chloride pretreatment against CC1 4 -induced lipoperoxidation and increases in serum enzymes . They attributed this protection to a cobaltous chloride induced decrease of cytochrome P-450 . The effects of the various atmospheres on the covalent binding of these compounds (Table 1) suggest that CHC1 3 is either metabolized differently than CC1 4 and CBrCl, or else the steady state levels of their reactive intermediates are affected differently by N2 or 02 . Regarding the latter point, the reactive intermediate(s) from CC1 4 may be inactivated by an 02 dependent process and those of CHC13 by a reductive process that is not OZ dependent . An atmosphere favoring increased decomposition of the reactive intermediates to inactive species would be observed as a reduction in binding . However, we favor the alternative proposal that the reduction in binding reflects different pathways of bioactivation . The reduction in binding of CHC1, by an atmosphere of N2 suggests that its bioactivation is mediated by a cytochrome P-450 oxidative pathway and that oxygen is probably incorporated into the reactive intermediate . This conclusion is further supported by the studies of Brown et al (31) who reported that in mice 80% of administered 14 CHC1, was recovered as 14 C02 " Although the identity of the intermediate is unknown, it could be trichloromethanol, which would decompose to phosgene (COC1 2 ) . Additional support for the classical cytochrome P-450 oxidative pathway in CHC1, bioactivation are the inhibitory effect of SKF 525-A on its binding (Table 1) and the seven fold increase in binding when liver microsomas are isolated from phenobarbital treated rats (9) . It is noteworthy that an atmosphere of N2 effects the bioactivation and/ or binding of CC1 4 , CHC1 3 and CBrCl, differently . Uehleke et al (32) reported that anaerobic conditions greatly enhanced the binding of CT4to liver endoplasmic reticulum proteins, as was observed in these studies . The enhanced binding of CC1 4 under N 2 suggests that CC1 4 is metabolized by cytochrome P-450 mediated reductive pathways and-that 02 inhibits biotransformation by competing for reducing equivalents . In many reductive pathways, SKF 525-A is known to be an ineffective inhibitor of drug metabolism and may actually increase the rate of reduction (23) possibly by increasing the rate of comsumption of trace amounts of oxygen remaining in the incubation mixtures . In this study SKF 525-A enhanced the binding of CBrCl, by 44% and was ineffective against CC1 4 . It was not possible to demonstrate an increase in binding of CBrCl, under N2, but all other aspects of its bioactivation and binding are similar to CC1 4 . The role of reduced glutathione in bioactivation and covalent binding of these three hepatotoxins remains unknown . It is extremely effective in decreasing the covalent binding . In phenobarbital treated animals CHC1,
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depletes liver glutathione presumably by the formation of a conjugate between the reactive intermediate (COC1 2 ) and glutathione (9,10) . However, it has not been possible to detect a decrease in the liver glutathione levels following administration of CC1 4 or CBrCl,(10) . Nevertheless, it seems possible that glutathione may decrease the levels of " CCl, by reducing it to chloroform and that the oxidized glutathione may be reduced back to glutahione by glutathione reductase . Whether this mechanism occurs in living animals, however, remains to be elucidated . References 1. 2. 3. 4. 5. 6. 7. 8. 9. 10 . 11 . 12 . 13 . 14 . 15 . 16 . 17 . 18 . 19 . 20 . 21 . 22 . 23 . 24 . 25 . 26 . 27 . 28 . 29 . 30 . 31 . 32 .
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