Chern.-BioL Interactions, 69 (1989) 217--224 217 Elsevier Scientific Publishers Ireland Ltd.

GLUTATHIONE AND L I P I D PEROXIDE LEVELS IN RAT LIVER FOLLOWING ADMINISTRATION OF M E T H A P Y R I L E N E AND ANALOGS

LIDIA HERNANDEZ and WILLIAM LIJINSKY

NCI-Frederick Cancer Research Facility, BRI-Basic Research Facility, Frederick, MD ~1701 fU.S.A./

(Received April 29th, 1988) (Revision received July 25th, 1988) (Accepted July 25th, 1988)

SUMMARY

The possibility was examined that the induction of tumors in rat liver by feeding methapyrilene, which is not mutagenic, is related to effects on gluta- thione levels and lipid peroxidation. Fischer 344 rats were given single-dose and multiple-dose t reatments with the anti-histamine methapyrilene (MP), which is carcinogenic in rats, and with two non-carcinogenic analogs, methafurylene (MF) and thenyldiamine (TD) and the effects on malonaldehyde (MDA) formation and glutathione (GSH) levels in the liver were investigated. After a single dose, MDA levels were increased at 6 h by MF and TD and at 24 h by MP. MDA levels returned to normal after 30 h with MP and MF, but not with TD. Levels of MDA (and other TBA-reactive products) after four daily t reatments were most elevated by TD, less elevated by MP, and were lowered by MF. Forty-two hours following t reatment with both MP and MF, MDA levels had returned to normal, but in TD-treated animals MDA remained high. GSH levels were highest after MF and MP, and remained high at 42 h, but TD induced only a small increase. There appears to be increased lipid peroxidation in the liver as a result of t rea tment of rats with MP, MF and TD. The greater response induced by TD, as well as the increased liver GSH levels after repeated administration of all three drugs indicate that lipid peroxidation in rat liver is not a particular effect related to the liver carcinogen methapyrilene.

K e y w o r d s : Methapyrilene -- Glutathione -- Lipid peroxidation -- Carcino- genesis - Rat liver

INTRODUCTION

The anti-histamine methapyrilene has been shown to induce t u m o r s in the

0009-2797/89/$03.50 © 1989 Elsevier Scientific Publishers Ireland Ltd. Printed and Published in Ireland

218

liver of F344 rats [1,2], but does not appear to be mutagenic [3,4] nor to have any detectable genotoxic effects [5]. Recent investigation has shown early ultrastructural changes in the liver of MP-treated rats, but not in rats t reated with non-carcinogenic analogs [6], and in rat liver epithelial cells treated in culture with MP [7], particularly mitochondrial proliferation and distortion, and the appearance of lamellar bodies in the cytoplasm. Increased early lipid peroxidation in the liver of MP-treated rats has also been reported [8].

It has been suggested that free-radical damage to DNA or cell membranes may be related to the induction of tumors [9,10]. This led us to investigate the role of lipid peroxidation in the action of methapyrilene. Many drugs are believed to exert their toxic effects on the liver by a mechanism involving glutathione (GSH) depletion or oxidation [11-14]. Sufficient GSH depletion may impair the effectiveness of GSH-dependent anti-lipoperoxidative systems for protecting against free radical-mediated damage that could potentially be initiated by reduced forms of oxygen released during microso- real or mitochondrial oxidative metabolism. Unchecked peroxidative damage can lead to membrane damage and/or possibly DNA damage. We examined early effects of methapyrilene in the livers of rats on GSH levels and lipid peroxidation rates, which might be related to the carcinogenicity of the drug. These changes could be compared with the corresponding effects in rats t reated with two non-carcinogenic analogs [15] of methapyrilene, metha- furylene and thenyldiamine (Fig. 1).

METHODS

Animal treatments Male F344 rats (10--12 weeks old) were obtained from the colony of the

Frederick Cancer Research Facility. Thenyldiamine and methafurylene were synthesized as previously described [15] and were dissolved in water by addi- tion of 10 N hydrochloric acid to pH 2 and a final concentration of 50 mg/ml. Methapyriline hydrochloride was obtained from Sigma Chemical Co. (St. Louis, MO) and dissolved in water at the above concentration. Animals received 0.4, 0.6 or 0.8 ml by gavage of these solutions corresponding to doses of 100, 150 or 200 mg/kg body wt., respectively. None of the treat- ments caused pathological changes in the liver detectable at 1 week after

/CH2CH2N~CH3 C~, . . CH3

Methapyrilene

~CH 3 /N / CH2CH2N ~" CH3

CH2[

Thenyldiamine

Fig. 1. Structures of MP, TD and MF.

3

C H~...... 3

Methalurylene

219

treatment. Control animals received equivalent volumes of water. All other chemicals were obtained from Sigma and Aldrich Chemical Co. (Milwaukee, WI).

Tissue preparation and biochemical determinations Rats were killed by decapitation at the various times shown following

t reatment and the livers were immediately removed. Approximately 2 g portions were weighed and immediately homogenized in 10 vol. of ice-cold 1.1% KC1 with a teflon pestle and glass homogenizer and a Wheaton overhead st irrer (Wheaton Instruments, Millville, N.J.}. Homogenates were immediately centrifuged at 9000 x g for 30 min at 4°C. Supernatant frac- tions ($9) were decanted into clean tubes at 4°C. For lipid peroxidation measurements, 0.2 ml aliquots of S9 were immediately incubated with 0.8 ml 20 mM sodium phosphate buffer (pH 7.0) for 2 h at 37 °C with gentle shaking, open to air; protein was precipitated with 0.2 ml each of saturated Ba(OH} 2 and ZnSO 4 solution and centrifuged [16]. The supernatant (0.5 ml) was added to 3 ml of HC1-KCI buffer (pH 2.0) and 1 ml of a 0.6% thiobarbituric acid aqueous solution, in a modification of the method of Uchiyama and Mihara [17]. The reaction mixtures were boiled for 30 min, cooled to 25°C and the absorbance measured at 535 nm in a Beckman Acta IV spectrophotometer. The absorbances were used to measure malonaldehyde (MDA) using a stand- ard curve prepared with 1,1,3,3-tetramethoxypropane. Another portion (0.45 ml) of each $9 sample was used to determine GSH content according to the procedure of Baars and Breimer [18] using 1-chloro-2,4-dinitrobenzene deriva- tives and analyzing the product by reverse-phase HPLC using a Waters model 660 programmer with a model 450 wavelength detector and a What- man ODS-2 reverse phase Partisil 10 R-8 column. Protein concentrations were determined by a micro modification of the method of Lowry [19]. All values reported represent mean _+ S.E. for three animals at each time point.

RESULTS

The effects on lipid peroxidation of a single administration of MP and its analogs at 100 mg/kg body weight are shown in Fig. 2. The levels of malonal- dehyde and other TBA-reactive products were 3.6-fold higher in the livers of MP-treated animals at 24 h, falling to control levels after 30 h. Both TD and MF increased MDA levels approximately 4.5 times, 6 h after treatment; the MDA levels remained elevated for at least 30 h in the case of TD, while MF treated animals had returned to control levels by that time.

To determine whether the drugs had a dose related effect on lipid peroxidation, rats were t reated with three different dose levels of MP and the effect on lipid peroxidation of liver $9 fractions incubated with and with- out added GSH was studied (Fig. 3). In $9 fractions from rats treated with 100 or 150 mg/kg body wt. of MP, lipid peroxidation increased 1.8- and 3.2- fold over controls by 24 h, respectively; this effect was not observed when $9 fractions were assayed in the presence of 0.5 mM GSH. $9 fractions from

220

c -

o Q. O} i=

i

o E C

<

24

22

20

18

16

14

12

10

8

-)$

2 6 10 14 18 22 26 30

Hours After Treatment

Fig. 2. Changes in MDA levels in extracts of liver of rats between 0 and 30 h after single treat- ment with 100 mg/kg of MP, MF or TD. Points represent the mean of 3 - 4 animals -+ S.E.; *P < 0.01.

32 t 28 t,,- I,, 20

o 16 E p .

12 p,,

8

F"~J l h [ ] l h + 500#M GSH • 24h

24h + 500#M GSH

0 100 150 200

Methapyrilene, mglkg Body Weight

Fig. 3. Effects of different doses of MP on the generation of MDA in extracts of liver prepared 1 h and 24 h after single treatment.

221

animals given 200 mg/kg body wt. MP showed only a 2-fold increase in MDA formation over controls, perhaps due to toxicity to the liver cells at that dose.

In view of the lack of relationship between the intensity and timing of GSH levels and capacity for lipid peroxidation following a single t reatment with the anti-histamines, the effects of several consecutive doses were inves- tigated, as in a chronic toxicity study, to determine whether repeated admin- istration of the drugs would produce different effects in the rat liver.

The same parameters were measured in animals which had received four consecutive 100 mg/kg daily doses of the drugs. Results in Figs. 4 and 5 show that, in MP-treated animals lipid peroxidation levels were higher than controls at 1 h and steadily decreased, leveling off at approximately 18 h after the last dose. TD-treated animals showed the same trend, but levels were higher and had not returned to control levels by 42 h. MF induced lipid peroxidation levels lower than controls for the duration of the experiment. TD-treated animals showed increased GSH levels above controls only at 42 h, while MP and MF-treated animals showed higher GSH levels than con- trols at earlier times. In TD-treated animals, GSH remained near control levels initially, and then rose by 42 h. MP- and MF-treated animals showed an initial increase which remained high throughout.

20 r o C 181- * * * * • MP

/ . M F

c

I I 1 I 1 I 1 l 0 6 12 18 24 30 36 42

H o u r s A f t e r T r e a t m e n t

Fig. 4. Changes in MDA levels in extracts of liver of rats between 0 and 42 h following 4 consec- utive dai ly d o s e s of 100 mg/kg of MP, MF or TD. Points represent the mean ± S.E. of 3-4 animals; * P < 0.05; * * P < 0.02; * * * P < 0.01.

222

200

160 C

e 120

E = 80

4 0 -

~k ~ * * *

* * *

oC AMP BMF • TD

I I I I I I I

0 6 12 18 24 30 36 42

Hours After Treatment

Fig. 5. Changes in GSH content in extracts of liver of ra ts between 0 and 42 h following 4 con- secutive daily doses of 100 mg/kg of MP, MF or TD. Points represent the mean _+ S.E. of 3 - 4 animals; *P < 0.05; **P < 0.02; ***P < 0.01.

DISCUSSION

There is an abundance of reports in the li terature describing increased lipid peroxidation in rat liver as a result of GSH depletion in vivo, in isolated primary hepatocyte cultures as well as in tissue homogenates [20--24]. This effect is a result of t reatment with some toxic agents [25,26], and it has been suggested that the membrane damage effected by the peroxidative response accounts for cell death or the tumor-promoting effects seen with some drugs [27,28].

It has been suggested that iron is normally protected in the cell by GSH [11], and in GSH-depleted cells iron would thus be able to stimulate iron- dependent lipid peroxidation. In some cases [29--31] this has been shown to be a mechanism of destruction of P-450 activity, resulting in suppression of metabolism and/or GSH conjugation of metabolites. In other cases [14], it appears that GSH levels have to reach a critical minimum in order for peroxidation to occur.

In our experiments a dose of MP was used which, when administered continuously in food, would induce tumors in F344 rats within 18 months [1]. Even at this level it seems clear that a single t reatment stimulated lipid peroxidation, but it is unclear whether this is related to GSH content. How- ever, GSH levels were not depleted and there were no significant changes with time (data not shown). Although added GSH decreased MP-induced lipid

223

peroxidation (Fig. 3) in vitro, this does not imply that protective mechanisms involving GSH-peroxidase are effective in vivo. Lipid peroxidation is not a unique effect of MP, compared with its two close analogs, although the inten- sity and timing of the effect is somewhat different. The two analogs induced, respectively, higher and lower responses than MP, which leads to the conclu- sion that induction of lipid peroxidation in rat liver is not related to induc- tion of liver tumors by methapyrilene.

ACKNOWLEDGEMENTS

This research was sponsored by the National Cancer Institute, DHHS, under contract No. Nol-C0-74101 with Bionetics Research Inc.

REFERENCES

1 W. Lijinsky, M.D. Reuber and B.N. Blaekwell, Liver tumors induced in rats by oral adminis- tration of the anti-histaminic methapyrilene hydrochloride, Science, 209 (1980) 817-819.

2 M. Oshima, J.M. Ward, L.M. Brennan and D.A. Creasia, Sequential study of methapyrilene hydrochloride-induced liver carcinogenesis in male F344 rats, J. Natl. Cancer Inst., 72 (1984) 759- 768.

3 A.W. Andrews, J.A. Fornwald and W. Lijinsky, Nitrosation and mutagenicity of some amine drugs, Toxicol. Appl. Pharmacol., 52 (1980) 237--244.

4 D.A. Caseiano and H.M. Schol, Methapyrilene is inactive in the hepatocyte-mediated Chinese hamster ovary/hypoxanthine-guanine phosphoribosyl transferase mutational assay, Cancer Lett., 21 (1984) 337- 341.

5 P.T. Iype, R. Ray-Chaudhuri, W. Lijinsky and S.P. Kelley, Inability of methapyrilene to induce sister cbromatid exchanges in vitro and in vivo, Cancer Res., 42 (1982) 4614-4618.

6 H.M. Reznik-Schiiller and W. Lijinsky, Ultrastructural changes in the liver of animals treated with methapyrilene and some analogs, Ecotoxicol. Environ. Safety, 6 (1982) 328-- 335.

7 P.T. Iype, C.D. Bucana and S.P. Kelley, Carcinogenesis by nonmutagenic chemicals: early response of rat liver cells induced by methapyrilene, Cancer Res., 45 (1985) 2184-2191.

8 M.I.R. Perera, S.L. Katyal and H. Shinozuka, Metbapyrilene induces membrane lipid peroxi- dation of rat liver cells, Carcinogenesis, 6 (1985) 925-927.

9 E. Tanaka, S. Hagino, T. Yoshida, Y. Kuroiwa, H. Ohno and H. Numata, The protective effect of cysteine on chemical-induced liver injury in rats, J. Toxicol. Sci., 9 (1984) 245--251.

10 L.S. Mulcahy and J.H. Gans, The fidelity of mouse liver mitochondrial DNA polymerase fol- lowing long-term administration of carbon tetrachloride, diethylnitrosamine, or phenobarbi- tal, MoL Pbarmacol., 24 (1983) 329--335.

11 A. Valenzuela, V. Fernandez and L.A. Videla, Hepatic and biliary levels of glutathione and lipid peroxides following iron overload in the rat: effect of simultaneous ethanol administra- tion, Toxieol. Appl. Pharmacol., 70 (1983) 87-95.

12 M. Dubin, S.G. Goijman and A.O.M. Stoppani, Effect of nitroheterocycllc drugs on lipid per- oxidation and glutathione content in rat liver extracts, Biochem. Pharmacol., 33 (1984) 3419

- 3423. 13 Y. Nakagawa, K. Tayama, T. Nakao and K. Hiraga, On the mechanism of butylated hydroxy-

toluene-induced hepatic toxicity in rats, Biochem. Pharmacol., 33 (1984) 2669--2674. 14 S.P. Srivastava, M. Das and P.K. Seth, Enhancement of lipid peroxidation in rat liver on

acute exposure to styrene and aerylamide a consequence of glutathione depletion, Chem.- Biol. Interact., 45 (1983) 373-380.

15 W. Lijinsky and R.M. Kovatch, Feeding studies of some analogs of the carcinogen methapyri- lene in F344 rats, J. Cancer Res. Clin. Oncol., 112 (1986) 57-60.

224

16 M. Somogyi, Determination of blood sugar, J. Biol. Chem., 160 (1945) 69-73. 17 M. Uchiyama and M. Mihara, Determination of malonaldehyde precursor in tissues by thio-

barbituric acid test, Anal. Biochem., 86 (1978) 271--278. 18 A.J. Baars and D.D. Breimer, Determination of giutathione in biological material by high

pressure liquid chromatography, Pharmaceutisch Weekblad Sci. Ed., 5 (1983) 145--148. 19 O.H. Lowry, N.J. Rosebrough, A.L. Farr and R.J. Randall, Protein measurement with the

folln phenol reagent, J. Biol. Chem., 193 (1951) 265-275. 20 E. Albano, G. Poll, A. Tomasi, A. Bini, V. Vannini and M.V. Dianzani, Toxicity of 1,2-

dibromoethane in isolated hepatocytes: role of lipid peroxidation, Chem.-Biol. Interact., 50 (1984) 255-- 265.

21 L.A. Videla, Chemically induced antioxidant-sensitive respiration. Relation to giutathione content and lipid peroxidation in the perfused rat liver, FEBS Lett., 178 (1984) 119--122.

22 M. Younes and C.P. Siegers, Formation of ethane in vitro by rat liver homogenates follow- ing glutathione depletion in vivo, Toxicol. Lett., 15 (1983) 213--218.

23 D.J. Ecobichon, Glutathione depletion and resynthesis in laboratory animals, Drug Chem. Toxieol., 7 (1984) 345--355.

24 M.Y.H. Farooqui and A.E. Ahmed, Circadian perodicity of tissue giutathione and its rela- tionship with lipid peroxidation in rats, Life Sci., 34 (1984) 2413--2418.

25 T. NoU and H. DeGroot, The critical steady-state hypoxic conditions in carbon tetrachloride- induced lipid peroxidation in rat liver microsomes, Bioebim. Biophys. Acta, 795 (1984) 356- 362.

26 E. Albano, G. Bellomo, R. Carini, F. Biasi, G. Poll and M.V. Dianzani, Mechanisms responsi- ble for carbon tetraehloride-indueed perturbation of mitochondrial calcium homeostasis, FEBS Lett., 192 (1985) 184-188.

27 D.J. Kornbrust and J.S. Bus, Glutathione depletion by methyl chloride and association with lipid peroxidation in mice and rats, Toxicol. Appl. Pharmacol., 72 (1984) 388-399.

28 Y. Ohno, K. Ormstad, D. Ross and S. Orrenius, Mechanism of allyl alcohol toxicity and pro- tective effects of low-molecular-weight thiols studied with isolated rat hepatocytes, Toxicol. AppL Pharmacol., 78 (1985) 169--179.

29 W.G. Levine, Glutathione, lipid peroxidation and regulation of eytochrome P450 activity, Life Sci., 31 (1982) 779--784.

30 M. Younes, M. Albrecht and C.P. Siegers, The role of iron in the NADPH-dependent lipid peroxidation due to giutathione depletion by phorone, Pharm. Res. Comm., 16 (1984) 153- 160.

31 R.L. Aft and G.C. Mueller, Degradation and covalent crosslinking of giutathione reductase by heroin, Life Sei., 36 (1985) 2153-2161.