Document 10vqoDoXNpxD7E1gGxpJMr1rX

XENOBIOTICA, 1976, VOL. 6, NO. 1U, 599-604 Effects of Aliphatic Chlorohydrocarbons on Drug-metabolizing Enzymes in Rat Liver in vivo HARRI VAINIO Department of Industrial Hygiene and Toxicology, Institute of Occupational Health, Helsinki, MAX G. PARKKI and JUKKA MARNIEMI Department of Physiology, University of Turku, Turku, Finland. {Received 17 January 1976) 1. Various polychlorinated hydrocarbons were administered iritragastrically to rats to examine their effects on the biotransformation capacity of the liver. Due to high toxicity, 1,1,2,2-tetrachIoroethane and pentachloroethane were given at a dose level equivalent to one quarter of that of CC14 and the other chlorohydrocarbons (i.e. 2-6 mmol /kg). 2. Carbon tetrachloride at 10-3 mmol/kg -was the most active in decreasing cytochrome P-450 content and the overall drug hydroxylation activities in rat liver. 1,1,2,2-Tetrachloroethane was the next most active in decreasing the hepatic drug oxidizing enzymic activities. 3. Epoxide hydratase activity in rat liver declined significantly after CCIt, 1,1,2,2-tetrachIoroethane and pentachloroethane administrations. 4. UDP-Glucuronosyltransferase was affected to a lesser extent than the microsomal mono-oxygenase or epoxide hydratase by chlorohydrocarbon treatment. [^Aduction 'Aliphatic chlorinated hydrocarbons have a widespread and essential role in :he chemical industry and in a variety of manufacturing operations. As a rroup, the chlorohydrocarbons share several biological properties. Some properties, such as toxicity are associated with only certain members of the series, and these manifestations may present a wide spectrum according to which compound is responsible for the poisoning. Plaa, Evans & Hine (1958) evaluated tepatotoxicity in mice by judging the impairment of detoxication of barbiturates ifter exposure of the mice to chlorinated hydrocarbons. More recently hepatic damage has been evaluated in mice by determination of the serum transaminase evels after exposure (Gehring, 1968). The binding of substrates to P-450 haemoprotein is supposed to be required or the compound to be metabolized by the mono-oxygenase system. The netabolism may result in either a decrease or an increase of biological activity; i.g. carbon tetrachloride is activated to its toxic form by microsomal monojxygenase system (cf. Recknagel, 1967). In a previous study we found that the iddition of various chlorinated hydrocarbons to liver microsomal suspension esulted in a type I spectral interaction (Pelkonen & Vainio, 1975). The in creasing number of chlorine atoms resulted in an increased magnitude of the ype I spectral change and an increased affinity to cytochrome P-450. To evaluate the relationship between the binding of chlorohydrocarbons to cyto chrome^ P-450 and their toxicity, the various chlorinated hydrocarbons were SL 037501 600 H. Vainio et al. administered intragastrically to rats and the activities of hepatic microsomalbound mono-oxygenase, epoxide hydratase and UDP-glucuronosyltransferase measured. Materials and methods Male Wistar rats weighing about 250 g fed ad libitum on commercial pellets (Hankkija Ltd., Turku, Finland) were used. Carbon tetrachloride (analytical grade, purity 99-8% (g.l.c.), Merck AG, Darmstadt, Germany) 10-3 mmol/kg, 1,1,1-trichloroethane (practical grade, purity at least 95% (g.l.c.) Fluka AG, Buchs, Switzerland) 10-3 mmol/kg, 1,1,2,2-tetrachloroethane (reagent grade, purity 98% (g.l.c.) Merck-Schuchardt AG, Munchen, Germany) 2-6 mmol/kg, tetrachloroethylene (technical grade, Tamro Ltd., Finland) 10-3 mmol/kg, pentachloroethane (practical grade, purity at least 95% (g.l.c.) Fluka AG, Buchs, Switzerland) 2-6 mmol/kg, and hexachloroethane (practical grade, purity at least 95% (g.l.c.) Fluka AG, Buchs, Switzerland) 10-3 mmol/kg were ad ministered intragastrically in olive oil 24 h before the rats were killed. Control animals were treated with an equivalent volume (5 ml /kg) of olive oil. CaCl2-precipitated microsomes were prepared as described earlier (Vainio & Aitio, 1974). Digitonin IE. Merck AG, Darmstadt, Germany) and trypsin (type XII, from bovine pancreas, Sigma Chemical Company, St. Louis, Mo, U.S.A.) treatments used in connection with UDP-glucuronosyltransferase activity determination were performed as previously described (Hanninen & Puukka, 1970; Vainio, 1973). Protein determination was carried out by the biuret method using bovine serum albumin (Armour Pharmaceuticals, Eastbourne, England) as reference (Layne, 1957). Aryl hydrocarbon hydroxylase (EC 1.14.14.2) was measured fluorometrically with an Aminco-Bowman spectrofluorometer using 3,4-benzpyrene as substrate (Nebert & Gelboin, 1968; Vainio, 1973). In determination of p-nitroanisole O-demethylase activity the formation of />-nitrophenol from p-nitroanisole was recorded with a Unicam SP-800 spectrophotometer (Netter & Seidel, 1964). The cytochrome P-450 content was measured according to the method of Omura and Sato (1964). NADPH-cytochrome c reductase activity was determined by monitoring the reduction of cytochrome c at 550 nm (Phillips & Langdon, 1962; Vainio & Hanninen, 1972). Epoxide hydratase activity was determined by the method of Oesch, Jerina & Daly (1971) as described earlier (Vainio & Parkki, 1974). The water phase containing styrene glycol formed was counted in Monophase 40 (Packard Instrument Co. Inc., Illinois, U.S.A.) without any further extraction with ethyl acetate. The counting efficiency achieved was 29% as determined by internal tritium standardization, (3H20 was a gift from Dr. Niilo Kaartinen, Department of Physiology, University of Turku). The tritiated styrene oxide (NEN Chemicals Ltd, Dreieichenhain, Germany) was treated as described earlier (Marniemi & Parkki, 1975) to remove soluble product formed during storage. UDP-Glucuronosyltransferase (EC 2.4.1.17) activity was determined with p-nitrophenol (0-5 mmol/1, Merck AG, Darmstadt, Germany) as aglycone. The UDP-glucuronic acid (Sigma Chemical Company) concentration used was 2-2 mmol/1 (Hanninen, 1968). SL 037502 Chlorohydrocarbons and Drug Metabolism SL 037503 Table 1. The effect of peroral administration of sevew^aliphatic chlorohydrocarbons on drug biotransformation P * enzymes in rat liver Compound NADPH-Cyt. c reductase (ftmol/g liver/min) Cyt. P-450 (nmol/g liver) 3,4-Benzpyrene p-Nitroanisole Epoxide hydratase hydroxylase O-demethylase (nmol/g liver/min) (nmol/g liver/min) (nmol/g liver/min) Oil Carbon tetrachloride 1,1,1-Trichloroethane 1,1,2,2-TetrachIoroethane Tetrachloroethylene Pentachloroethane Hexachloroethane 0-55 + 0-06 0-41+0-06 0-57 + 0-05 0-43+0-11 0-63 + 009 0-61 +0-05 0-73 0-05 f6) 6-95 0-54 (6) 187114 (6) (5) 1-47 0-39*** (S) 113 + 7*** (S) (5) 3-93 0-50** (5) 138114* (5) (4) 3-700-74** (4) 93-0112** (4) (5) 5-8110-61 (S) 156119 (5) (5) 4-54 + 0-56* (S) 124 + 8** (5) (5) 3-38 + 0-73** (5) 184+12 (5) 2-20 + 0-20 (7) 10-610-89 (6) 0-76+ 0-13*** (6) 3-111-5** (S) 1-9610-21 (6) 7-5 i 1-7 (5) 0-6510-24** (4) 4-611-5** (5) 1-2510-12** (5) 7-2+1-1* (4) 1-5610-27 (5) 7-710-6 (5) 0-7510-07*** (5) 5-910-5** (5) The dose given was 2-6 mmol/kg body wt. for 1,1,2,2-tetrachloroethane and pentachloroethane, whereas that for other halogenated hydrocarbons was 10*3 mmol/kg body wt. The activities of NADPH-cytochrome c reductase (cytochrome c reduced, ;umol/g liver w.wt./min), epoxide hydratase (styrene glycol formed, nmol/g w.wt./min), aryl hydroxylase (activity expressed as nmol hydroxylated benzpyrene formed/g liver w.wt./min), p-nitroanisole O-demethylase (p-nitroanisole formed/nmol/g liver w.wt./min) as well as the amount of cytochrome P-450 (nmol/g liver w.wt.) were measured. The number of experiments is given in parentheses. Statistical analysis as compared to oil controls was performed with Student's t-test (**: 2P < 0-001; **: 2P<0-01; *-. 2P<0-05). K) O' o 602 H. Vainio et al. Results Various chlorinated hydrocarbons were administered intragastrically to rats in equivalent doses (10-3 mmol/kg body wt.) except 1,1,2,2-tetrachloroethane and pentachloroethane, which were given at a dose level of 2-6 mmol/kg body wt. due to the lethality of higher doses to rats. Of the component reactions of the microsomal mono-oxygenase system, NADPH-cytochrome c reductase was not significantly affected by the chlorohydrocarbons (Table 1). Cytochrome P-450 was, however, greatly influenced by the treatments. Carbon tetrachloride, especially, was active in decreasing the P-450 haemoprotein in liver microsomes (cf. Recknagel, 1967). The other chlorinated hydrocarbons, at the dosage used, caused a diminution of cytochrome P-450 concentration to about one half of the control level, except for tetrachloroethylene which caused only a slight decrease (18%) (Table 1). The activities of overall drug oxidation reactions; hydroxylation of 3,4-benzpyrene and O-demethylation of p-nitroanisole were decreased considerably especially by carbon tetrachloride, 1,1,2,2tetrachloroethane, and hexachloroethane treatments (Table-1). Epoxide hydratase activity, which is responsible for conversion of the inter mediate epoxides into dihydrodiols (Oesch, 1973), declined greatly after carbon tetrachloride (40%), 1,1,2,2-tetrachloroethane (50%) and pentachloroethane (34%) administration. Statistically a significant decrease was also achieved by 1,1,1-trichloroethane. The other compounds tested had no significant effect on this microsomal-bound enzyme (Table 1). UDP-Glucuronosyltransferase activity, when measured from intact ' native ' microsomes, was increased (53%) by carbon tetrachloride pretreatment of rats (Table 2). If the microsomes had been pre-treated in vitro with digitonin, a Table 2. The effect of peroral administration of several aliphatic chlorohydro carbons on UDP-glucuronosyltransferase activity. Compound Native microsomes Digitonin-treated microsomes Trypsin-treated microsomes Oil Carbon tetra chloride 1,1,1-Trichloroethane 1,1,2,2-TetrachIoroethane Tetrachloroethylene Pentachloro ethane Hexachloro ethane 8-53 1-25 (6) 13-01 1-90 (5) 6-02 1-37 (5) 6-42 + 0-73 (4) 8-02 1-60 (5) 9-11 1-47 (5) 4-91 0-54* (5) 72-0 2-30 (6) 60-6 3-3 # (5) 72-8 6-5 (5) 60-ll-8#* (4) 64-3 4-4 (5) 67-9 2-2 (5) 69-9 3-3 (5) 13-62-5 (6) 13-6 1-3 (5) 14-1 2-5 (5) 11-0 2-6 (4) 22-2 8-2 (5) 15-5 + 1-8 (5) 16-5 5-0 (5) The activity was measured both in native, in digitonin- and in trypsin-activated microsomes and was expressed as nmol p-nitrophenol bound/g liver w.wt./min in rat liver. For other details, see Table 1. SL 037504 Chlorohydroearbom and Drug Metabolism 603 se in measurable UDP-glucuronosyltransferase activity could be observed * treated with carbon tetrachloride or tetrachloroethane (about 16% in ases). After trypsin digestion of the hepatic microsomes in vitro, the ransferase activity was at the control level even in carbon tetrachloride pre dated rats (Table 2). The other chlorohydrocarbons studied had only minor ffects on microsomal UDP-glucuronosyltransferase, both when measured in native * or in ` activated ' microsomes (Table 2). liscussion In general, the effects which various chlorinated hydrocarbons have on the ctivities of different microsomal drug-metabolizing enzymes seem, to have no orrelation to the previously observed affinity towards cytochrome P-450 Pelkonen & Vainio, 1975). The effects at the subcellular level are obviously more dependent on specific metabolism of the chlorohydrocarbons than mere binding to P-450 haemoprotein. Cytochrome P-450, in addition to its catalytic role as terminal oxygen activator in drug metabolism, also functions as an active trapping agent for certain foreign compounds (Grundin et al.t 1974). This means that compounds .'hich have a high affinity towards P-450 haemoprotein are effectively extracted rom blood into tissues such as liver and kidney. In some instances even more oxic derivatives than the parent compound are formed during oxidation by ytochrome P-450. A good example here is carbon tetrachloride, the active orm of which is formed by the microsomal mono-oxygenase system and which iter sparks a process of lipid peroxidation (Slater, 1966; Recknagel, 1967). early destruction of a component of the liver endoplasmic reticulum might to either direct attack of free radicals arising from carbon tetrachloride r ttfthe deleterious effect of the lipid peroxidation process (Recknagel fit Glende, 973; Vainio & Parkki, 1974). While destroying the structure of the microsomal Vembranes, cytochrome P-450 is converted to inactive P-420 form and conequently the drug oxidations are inhibited. However, UDP-glucuronosylransferase, which resides in a deeper location in the membrane structure (for eferences, see Vainio, 1975), is only activated in vivo by carbon tetrachloride re-treatment of rats (cf. also Aitio, 1974). The other chlorinated hydroarbons now studied had a distinct decreasing effect on microsomal mono xygenation, whereas UDP-glucuronosyltransferase was not much affected by lem. 1,1,2,2-Tetrachloroethane and pentachloroethane could not be given at a osage level equivalent to carbon tetrachloride due to their lethality, but had ) be decreased to one fourth of that dose. However, compounds with even till higher chlorine content, e.g. hexachloroethane, which has a high affinity Dwards cytochrome P-450 (Pelkonen & Vainio, 1975), was well tolerated by rats, 'etrachloroethane, which was administered at a dose level of one fourth of itrachloroethylene, was surprisingly active in destroying the metabolic functions f microsomes. On the other hand, the metabolism of tetrachloroethylene as ell as ethenes generally have been suggested to proceed via epoxides (Leibman ; Ortiz, 1970). Epoxides have been implicated in various toxic reactions such s carcinogenesis, mutagenesis and necrogenesis (Jerina & Daly, 1974). It is SL 037505 604 Chlorohydrocarbons and Drug Metabolism possible, however, lhal the biotransformation of tetrachloroethane proceeds via formation of a radical similarly to carbon tetrachloride (cf. Recknagel & Glende, 1973). The actions of carbon tetrachloride and tetrachloroethane in the present study differed, however, in various aspects, e.g. in behaviour towards UDP-glucuronosyltransferase. Epoxide hydratase and cytochrome P-450 are possibly intimately linked in the microsomal membrane (Oesch & Daly, 1972). In the present study it was found that epoxide hydratase activity was decreased by administering many chlorinated hydrocarbons to rats, e.g. by carbon tetrachloride, 1,1,2,2-tetrachloroethane and pentachloroethane. It is interesting to note that among the compounds studied tetrachloro- and pentachloroethane were also the most toxic derivatives. This may be suggestive of their possible metabolic conversion to toxic epoxide derivatives. Some differences in the behaviour of cytochrome P-450 and epoxide hydratase could be seen, e.g. hexachloroethane, which decreased the content of cytochrome P-450 to about one half of the original (and highly inhibited the hydroxylation reactions) had no effect on epoxide hydratase activity. This may be due to the high affinity of this compound towards cytochrome P-450 as observed earlier (Pelkonen & Vainio, 1975). Acknowledgment This study has been supported by grant from U.S. Public Health Service (AM-06018). References Aitio, A. (1974). FEBS Lett., 42, 46. Gehring, P. J. (1968). Toxic, appl. Pharmac., 13, 287. Grundin, R., Moldeus, P., Orrenics, S., Borg, K. O., SkAnbbrg, I. & von Bahr, C. (1974). Acta Pharmac. Tox., 35, 242. HAnninen, O. (1968). Ann. Acad. Sci. Fenn. Ser. A II, 142, 1. HAnninen, O. & Puukka, R. (1970). Suomen Kemistilehti, 43, 451. Jerina, D. M. & Daly, J. W. (1974). Science, 18S, 573. Layne, E. (1957). In Methods in Enzymology (Colowick S. P, & Kaplan, N. O., eds.), Vol. 3, p. 447. New York: Academic Press. Leibman, K. C. Sc Ortiz, E. (1970). J. Pharmac. exp. Ther., 173, 242, Marniemi, J. & Parkki, M. G. (1975). Biochem, Pharmac., 24, 1569. Nebert, D. W. & Gelboin, H. V. (1968). J. biol. Chem., 234, 6242. Netter, K. J. & Seidel, G. (1964). J. Pharmac. exp. Ther., 146, 61. Oesch, F. (1973). Xenobiotica, 3, 305. Oesch, F. & Daly, J. (1972). Biochem. Biophys. Res. Commun,, 46,1713. Oesch, F., Jerina, D. M. & Daly, J. (1971). Bioehim. Biophys. Acta, 227, 685. Omura, T. & Sato, R. (1964). J. biol. Chem., 239, 2379. . Pelkonen, O. & Vainio, H. (1975). FEBS Lett., 51,11. Phillips, A-. H. & Langdon, R. G. (1962). jf. biol. Chem., 237, 2652. Plaa, G. L., Evans, E. A. & Hine, C. H. (1958). J. Pharmac. exp. Ther., 123, 224. Recknacel, R. O. (1967). Pharmac. Rev., 19, 145. Recknagel, R. C. & Glende, E. A. Jr. (1973). CRC Critical Revieas in Toxicology, 2, 263. Slater, T. F. (1966). Nature (Lond.), 209, 36. Vainio, H. (1973). Xenobiotica, 3, 715. Vainio, H. (1975). Proceedings of the Sixth International Congress of Pharmacology, Helsinki, Finland, vol. 6, pp. 53-66. Vainio, H. & Aitio, A. (1974). Acta Pharmac. Tox., 34, 130. Vainio, H. & HAnninen, O. (1972). Suomen Kemistilehti, 45, 56. Vainio, H. & Parkki, M. G. (1974). Res. Commun. Chem. Path. Pharmac., 9, 511. SL 037506