Covalent binding of halogenated volatile solvents to subcellular macromolecules in hepatocytes

Life Sciences, Vol. 29, pp. 1207-1212 Printed in the U.S.A.

Pergamon Pres

COVALENT BINDING OF HALOGENATED VOLATILE SOLVENTS TO SUBCELLULAR MACROMOLECULES IN HEPATOCYTES Michael L. Cunningham,

A. Jay Gandolfi,

Klaus Brendel and I. Glenn Sipes

Department of Pharmacology and Toxicology Arizona Health Sciences Center, Tucson, Arizona 85724 (Received in final form July 9, 1981) Summary Rat liver hepatocytes were used to bioactivate the aliphatic halides-carbon tetrachloride, trichloroethylene, and methylene chloride. Optimum bioactivation occurred under a nitrogen atmosphere for carbon tetrachloride and under an oxygen atmosphere for trichloroethylene and methylene chloride. All were found to alkylate lipid and protein, while only carbon tetrachloride and trichloroethylene alkylated DNA and RNA. Introduction The liver is the major organ for the metabolism of xenobiotics (i). Therefore, isolated hepatocytes would make an excellent model for the study of drug metabolism, as evidenced by the increasing use of the cells in biotransformation studies (2,3). This system offers the advantages of maintaining the integrity of the perfused liver system, the conducting of many parallel experiments with samples of cells from one liver, the examination of phase I and phase II drug metabolism in their in situ architectural framework (2), and the ability of manipulation of various parameters such as glutathione levels, oxygen concentration, or cytochrome P-450 levels (4,5). As is demonstrated in this study, the metabolism and bioactivation of volatile compounds, partitioned between the gas and liquid phases of the incubation, can be studied in isolated hepatocytes. The compounds are very difficult to work with using whole animals or liver perfusions and the cost of exposing whole animals to radiolabeled volatile compounds is often prohibitive. In this study, carbon tetrachloride (CC14) was bioactivated to reactive intermediates which covalently bound to tissue macromolecules. The bioactivation of CCI 4 was greatly enhanced in the absence of oxygen whereas the bioactivation of trichloroethylene (TCE)* and methylene chloride (MC)* required oxygen. Interestingly, the hepatocytes remain viable during the incubation in a low oxygen atmosphere and retain their biotransformation ability. Finally, covalent binding to DNA and RNA in the hepatocytes was found only with TCE and CC14, which are known hepatocarcinogens (6), and not with MC°

Abbreviations:

trichloroethylene

(TCE); methylene chloride

0024-3205/81/121207-05502.00/0 Copyright (c) 1981 Pergamon Press Ltd.

(MC).

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Materials and Methods Hepatocyte Preparation: Male Sprague-Dawley rats (200-500g) were obtained from Hilltop Laboratories (Chatsworth, CA). Hepatocytes were prepared by collagenase perfusion of ex situ rat liver (7). Calcium free buffer was perfused through the portal vein for 5 min. followed by the addition of 0.05% (w/v) collagenase (Worthington type II) and calcium chloride to 5 mM. After 15 min. the cells were sieved through 60 micron nylon mesh, washed twice with buffer and resuspended in Gibco Medium 199 with 10% fetal calf serum to 8x107 cells/ml. Viability was determined by trypan blue exclusion and only preparations with 85% or greater viability were used. Incubation and Macromolecule Isolation: Five ml of hepatocyte suspension were placed into 25 ml Erlenmeyer flasks, the headspace was exchanged five times with 02 or N 2 to obtain the desired atmosphere, capped and incubated at 37°C in a gyratory shaker. The 14C-labeled aliphatic halides (New England Nuclear) were 99% pure as determined by the supplier. The radiolabeled organohalogens were administered undiluted to attain the lowest substrate concentrations possible and to prevent cytotoxicity and enzyme leakage (8). The specific activities were 53.0, 5.0 and 3.9 mCi/mmole for CC14, TCE, and MC, respectively. The incubations were initiated by injection of 2 ~Ci of the 14C labeled substrate into each flask through Teflon septums resulting in substrate concentrations of i, 14 and 42 ~M for CC14, TCE, and MC, respectively. At various time intervals 0.6 ml was removed for isolation and exhaustive solvent extraction of proteins and lipids. DNA and RNA were isolated from the remaining cells after their lysis by the addition of 5 ml 2% sodium dodecyl sulfate. Following three i hour extractions of the lysed cells with chloroform:isoamyl alcohol (10:2 v/v), nucleic acids were precipitated with i0 ml 0.4 N perchloric acid. After centrifugation, the pellets were solubilized in 2.0 ml 0.1M Tris-HCl; i raM EDTA, pH 7.4 and incubated 2 hours at 37°C with 50 pg ribonuclease (boiled 30 minutes to destroy deoxyribonuclease activity). Four ml 0.4 N perchloric acid were added and ribonucleotides were recovered in the supernatant after centrifugation. The pellet was solubilized in 2°0 ml 0.i M Tris-HCl pH 7.4 containing 2 mM MgCI 2 and incubated with 50 ~g deoxyribonuclease I for 2 hours at 37°C. Four ml of 0.4 N perchloric acid were added and deoxyribonucleotides were recovered in the supernatant after centrifugation. Macromolecule Quantification: Total lipids were quantified gravimetrically (i0) and protein content determined by the Coomassie dye binding technique (ii). Nucleotides were quantified by measuring the absorbance at 260 nm (12). Contamination of RNA in DNA was determined by the orcinol reaction (13) and DNA in RNA was determined by the diphenylamine reaction (14). Protein contamination in nucleotide fractions was assayed by the Coomassie technique (ii). Results and Discussion These studies demonstrated that isolated rat hepatocytes are useful in examining the bioactivation of volatile halogenated hydrocarbons. They were capable of producing reactive intermediates from three aliphatic halides that resulteg in the alkylation of the macromolecules of the hepatocyte. Hepatocytes offer the advantages of duplicating the morphology of in situ liver cells with respect to the bioactivation enzymes and subcellular organelles (2). Also, the relative contributions of microsomal activating enzymes and cytosolic conjugation enzymes to the bioactivation of xenobiotics can be assessed.

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Substrate concentrations were kept as low as possible in order to study the bioactivation of these compounds without cytotoxicity and loss of cell viability (8). This was done by using the radiolabeled compounds undiluted and using ethanol as the vehicle.

TABLE I Factors Mediating the Binding of Volatile Xenobiotics and Protein in Isolated Rat Hepatocytes.

Substrate

Atmosphere a (02 / N2) Protein

Lipid

GSH Depletion b

to Lipid

Phenobarbital c Induction

Protein

Lipid

Protein

Lipid

CCI 4

0.09

0.06

100%

100%

145%

120%

TCE

9.63

5.08

74%

74%

288%

238%

MC

8.53

11.96

44%

38%

44%

40%

Ratio of covalent binding observed under oxygen and nitrogen atmosphere. Values are the mean of 3-6 determinations for each condition. 30 minutes prior to hepatocyte isolation rats were treated IP with 0.6 ml/kg of diethylmaleate. Values are expressed as the mean percent of binding observed in hepatocytes from untreated rats with N=5. Rats were pretreatedwith 3 consecutive daily doses of phenobarbital (80 mg/kg, IP) beginning 4 days before isolation of hepatocytes. Values are expressed as the mean percent of binding observed in hepatocytes from untreated rats with N=5.

Isolated rat hepatocytes were also shown to be capable of bioactivation in the absence of oxygen. The covalent binding of CCI 4 equivalents to macromolecules was greatly enhanced in the absence of oxygen and was almost eliminated in the presence of oxygen (Table i). Cell viability, which was not affected by the low substrate concentrations used in this study, did decline after 30 minutes of anaerobic incubation, regardless of substrate. The bioaetivation of TCE and MC was enhanced in the presence of (Table I). This is consistent with the proposed oxidative metabolism to an epoxide (15) and MC to an acyl chloride (16). Viability could maintained in hepatocytes incubated with oxygen and TCE or MC for up hours (data not shown).

oxygen of TCE be to four

Hepatocytes from rats pretreated with phenobarbital to increase eytochrome P-450 levels showed increased binding of CCI 4 and TCE to lipids and proteins but decreased the binding of MC to these macromolecules. CC14 and TCE are known to be bioactivated by the microsomal mixed function oxidase system (15,17). Methylene chloride however, is also bioactivated by the phase II enzyme systems (16).

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N2 Atmosphere CCI4 00-

5C

ET)

E o0

.~

o(

500-

02 Atmosphere TCE <

400-

o--o RNA = DNA ~ - ~ Lipid = =Protein

/

20010000

'

10

T- 2'0 ..... '---~

50-

~ r

0

10

r

15

l

20

l

30

Incubation Time (min) Fi$. 1

Effect of incubation time on the covalent bindin Z of CC14, TCE and MC to macromoleeules in hepatoeytes. Values are the mean of three separate de terminat ions,

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Hepatocellular glutathione depletion with diethylmaleate decreased TCE and MC binding to lipid and protein (Table I). TCE may be bioactivated by cytochrome P-450 to an epozide or possibly by reaction with glutathione to produce the potent alkylating species dichlorovinyl cysteine (18). Decreased GSH would decrease the contribution of this reactive species to the total binding observed for TCE. The phase II biotransformation of MC has been postulated to involve glutathione catalytically and produce a S-chloromethyl glutathione intermediate (16). This is similar to the reactive bis-halomethyl ethers and may be the metabolite primarily responsible for covalent binding of MC in the hepatocytes system. Thus, depletion of hepatocellular glutathione content by diethylmaleate would inhibit this pathway and result in decreased binding. It is not surprising that GSH depletion had little effect upon CCI 4 bioactivation. CCI 4 does not significantly deplete hepatocellular glutathione, although one of the reactive intermediates of CC14, carbonyl chloride, does react with glutathione (19,20). It should also be stressed that diethylmaleate may inhibit cytochrome P-450 mediated reactions, so it may be difficult to interpret the effects of this agent in an integrated system such as isolated hepatocytes (21,22). CCI 4 and TCE metabolites were found to alkylate RNA and DNA whereas MC metabolites did not (Figure i). CCI 4 and TCE have been reported to be carcinogenic (6,23) and alkylation of nucleic acids has been postulated to be a mechanism whereby chemicals induce carcinogenesis (24). MC did not bind to hepatocyte DNA under any conditions (Table I). This correlates with the finding that prolonged MC exposure does not result in increased frequency of tumor formation in rats (25).

Acknowledgement This research was supported by NIH Grant CA 21820.

References i. 2. 3. 4. 5. 6. 7. 8. 9. i0. ii. 12. 13. 14. 15. 16.

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