Comparison of in vivo and in vitro methods for assessing the effects of carbon tetrachloride on the hepatic drug-metabolizing enzyme system

Toxicology Letters, 42 (1988) 309-316

309

Elsevier

TXL 02014

Comparison of in vivo and in vitro methods for assessing the effects of carbon tetrachloride on the hepatic drug-metabolizing enzyme system Robert W. Chadwick’, M. Frank Copeland’, Bruce A. Trela2 and Bernard M. Most3 ‘Health Effects Research Laboratory,

Environmental

Research Center, (MD-681, U.S. Environmental

Protection Agency, Research Triangle Park, NC2771 I, U.S.A., icology, School of Pharmacy and Pharmacal Sciences, Purdue U.S.A.,

Gary P. Carlson2,

‘Department of Pharmacology and ToxUniversity, West Lafayette, IN 47907,

and 3Northrop Services, Research Triangle Park, NC 27709, U.S.A.

(Received 20 April 1988) (Accepted 2 May 1988)

Key words: Model substrate assay; Phase 1 reaction; Phase II reaction; Liver; Urinary metabolite; Lin-

dane; r-Hexachlorocyclohexane;

Carbon tetrachloride

SUMMARY The effect of a single i.p. injection of 0, 20, 200, and 1000 @kg carbon tetrachloride on the activity of the hepatic drug-metabolizing enzyme system was measured in the rat by a model substrate assay employing lindane (y-hexachlorocyclohexane) and by a battery of in vitro enzyme assays. The data in this study indicated that carbon tetrachloride had a biphasic influence on the phase I reactions with the lowest dose inducing a significant increase in enzyme activity while the highest dose produced significant inhibition. Significant CCla-induced reductions in glucuronyltransferase and sulfotransferase activities were also observed while the effect on glutathione-S-transferase was ambiguous. The in vivo and in vitro assays showed good agreement.

Address for correspondence: Dr. Robert W. Chadwick, Health Effects Research Laboratory, Environmental Research Center, (MD-68), U.S. Environmental Protection Agency, Research Triangle Park, NC 27711, U.S.A. Abbreviations: -r-HCH, y-hexachlorocyclohexane (lindane); EGS, ethylene glycol succinate; PPO, 2,5-diphenyloxazole; POPOP, 1,4-bis-2-(5phenoxazolyl)-benzene. The research described in this article has been reviewed by the Health Effects Research Laboratory, U.S. Environmental Protection Agency and approved for publication. Approval does not signify that the contents necessarily reflect the views and policies of the Agency nor does mention of trade names or commercial products constitute endorsement or recommendation for use.

310

INTRODUCTION

Recently the effects of ally1 alcohol, ethanol, phenobarbital, mercuric chloride and bromobenzene on the hepatic drug-metabolizing enzyme system have been determined by both in vitro and in vivo methods [l-5]. There was excellent agreement, both qualitatively and quantitatively, in the results from both methods. These data indicated that metabolism of the model substrate y-hexachlorocyclohexane (yHCH, lindane) provided a satisfactory in vivo index to chemically induced alterations in both phase I and phase II reactions. The work reported here is part of an ongoing effort to evaluate the use of the model substrate assay, employing lindane, to provide an in vivo index of chemically induced perturbations in hepatic phase I and phase II metabolism. The effect of a single i.p. injection of 0, 20, 200, or 1000 pi/kg of the hepatotoxicant carbon tetrachloride (CCL) on both phase I and phase II reactions was determined by the model substrate assay and compared to a battery of in vitro measurements. MATERIALS

AND

METHODS

Weanling female Fischer 344 rats, obtained from the Charles River Breeding Laboratory (Kingston, NY) were housed individually in stainless steel cages both at Purdue University, where the in vitro assays were conducted, and at the E.P.A. Environmental Research Center, where the model substrate assay was employed. The rats had free access to Purina lab meal and water. Light was maintained on a 12:12 light/dark cycle and room temperature at 22 f 2°C. Bed-O-Cobs (The Andersons Cob Division, Delphi, IN) was used as bedding in both laboratories. Two weeks after the animals were received, four groups of six rats were dosed i.p. with 0, 20, 200, or 1000 pi/kg carbon tetrachloride (Mallinckrodt, Paris, KY). The vehicle was peanut oil and dosing volume was 0.1% of the body weight of the animal. Twenty-four hours after treatment with CCL, all rats were dosed (s.c.) with 19.7 mg lindane (containing 3.8 &i [U-14C]lindane)/kg for the in vivo assay. The [U-‘4C]lindane, with a specific activity of 54 mCi/mmol, and a radiochemical purity of 98% was obtained from Amersham, Arlington Heights, IL. Following treatment with lindane, the animals were transferred to animal containment chambers (PlasLabs, Lansing, MI) where urine, feces and expired air were collected for 24 h. Urinary lindane metabolites were extracted and analyzed as previously reported [3]. Urine extracts were analyzed for lindane metabolites on a Tracer Model 220 gas chromatograph equipped with a 63Ni electron-capture detector and linearizer. The column consisted of a 183 x 0.63 cm O.D. glass U-tube packed with 5% EGS f 1% HsP04 on 90/100 mesh Anakrom A (Analabs, North Haven, CT) and was maintained isothermally at 170°C. The 5% methane/argon carrier gas flow rate was regulated at 55 cm3/min. Radioactivity was analyzed in a Packard Tri-Carb

311

Model 3380 liquid scintillation spectrometer using a quench correction curve and a scintillation solution containing 500 ml toluene, 500 ml cellosolve, 5.0 g 2,Sdiphenyloxazole (PPO), and 50 mg 1,4-bis-2-(5-phenoxazolyl)-benzene (POPOP). For the in vitro work, 24 h after dosing with CCld, whole liver homogenates, microsomal and cytosolic fractions were prepared for determination of sulfotransferase [6], glucuronyltransferase [7], ethylmorphine demethylase [8], aldrin epoxidase [9], and glutathione-S-transferase [lo]. Proteins were quantified using the method of Lowry et al. [l 11. Duncan’s multiple range test [12] was used as an aid in the interpretation of the data from this study. Comparisons were considered significantly different at P< 0.05. RESULTS

Administration of carbon tetrachloride appeared to have a biphasic effect on the phase I reactions when measured either in vitro (Table I) or in vivo (Table II), For example, while the low dose significantly induced ethylmorphine demethylase activity in vitro, the high dose inhibited activity to levels less than one-third that of the control (Table I). Similarly, the low dose of CC4 increased the formation and excretion of 2,4,6-trichlorophenol and the total alcohol metabolites of lindane in vivo, whereas the high dose produced significant reductions in the excretion of 2,4,6-trichlorophenol, 2,4,5-trichlorophenol and 2,3,4,6-tetrachlorophenol (Table II). The oxidative degradation of lindane to the alcohol metabolites, 2,4,6-trichlorophenol and 2,3,4,6-tetrachlorophenol, and the dehydrochlorination of lindane to 2,4,5-trichlorophenol represent separate and distinctly different phase TABLE I EFFECT OF CARBON TETRACHLORIDE

ON PHASE I REACTIONS IN VITRO

Data are means + SEM of six rats. Treatmenta

Ethylmorphine demethylaseb

Alirin epoxidation’

Aldrin epoxidationd

Control 20 /J/kg 200 PI/kg 1000 $/kg

1.83 2.25 0.92 0.53

0.041 0.056 0.050 0.029

2.35 2.86 2.63 I.33

& + f +

0.04 0.12* 0.05* 0.04*

z!z0.004 * 0.009 + 0.006 + 0.005

+ 0.14 + 0.38 of: 0.29 f 0.27*

a Carbon tetrachloride was administered i.p. to groups of six rats. Controls received peanut oil (vehicle). Rats were sacrificed 24 h after treatment. b nmol HCHO/mg protein/min. ’ nmol/mg protein/min. d nmol/rat/min. * Significantly different from control (P
312

TABLE

11

EFFECT

OF CARBON

Data are means Treatmenta

TETRACHLORIDE

ON PHASE

I REACTIONS

+ SEM of six rats. Total

alcohol

Total

excretionb

2,4,6-

Total

excretionb

excretionb

excretionb

0.94

0.40

* 0.022

1.66 t

0.75

+ 0.09*

2.11

200 al/kg

0.64

+ 0.06*

1.20 * 0.11

0.68

1000 pi/kg

0.28

f

0.55

0.24

tetrachloride

b Percent

administered

* Significantly

different

2,4,5-

trichlorophenol

0.41

Rats were sacrificed

Total

tetrachlorophenol

20 al/kg

0.08

2,3,4,6-

trichlorophenol

Control

a Carbon

IN VIVO

was administered

0.17

& 0.25* f

0.22*

i.p. to groups

+ 0.06

1.13 f

0.135

+ 0.03

0.44

* 0.05

+ 0.07

0.30

& 0.03

+ 0.09*

0.14

+ 0.04*

of six rats. Controls

received

peanut

oil (vehicle).

24 h after treatment. dose/24

h.

from

control

(P
I reactions [13-161. Inhibition was observed in three of the four phase I reactions measured in vivo (Table II). Only the excretion of the alcohols was not significantly reduced. Phase II reactions were also affected by carbon tetrachloride, whether measured in vitro (Table III) or in vivo (Table IV). Glucuronyltransferase activity, with naphthol and chloramphenicol as substrates, was significantly inhibited by the two higher doses of carbon tetrachloride (Table III). The high dose also significantly inhibited sulfotransferase in vitro but only when the data were expressed in terms of activity per whole liver. Only one of the two glutathione-S-transferase substrates, 1,2-dichloro-4-nitrobenzene, responded to carbon tetrachloride treatment with significantly reduced conjugation at the high dose (Table III). When the effect of CCL on glucuronyltransferase was determined in vivo, a significant 55% reduction in the excretion of lindane-derived glucuronides indicated inhibition at the high dose. Similarly, carbon tetrachloride-induced inhibition of sulfotransferase was suggested by the 65% decrease in the excretion of lindane-derived sulfates (Table IV). However, because the proportion of the metabolites conjugated as glucuronides and sulfates was not also significantly reduced, the carbon tetrachloride-induced inhibition of glucuronyltransferase and sulfotransferase in vivo is somewhat qualified. Carbon tetrachloride had no significant effect on glutathione-S-transferase as measured by the excretion of lindane-derived mercapturic acids (Table IV). DISCUSSION

The dose-response effect of 0, 20, 200, or 1000 pi/kg carbon tetrachloride on the drug-metabolizing enzyme system was determined by a model substrate assay and a battery of in vitro enzyme assays. There was good agreement between the in vivo and in vitro results for the phase I reactions, with an indication that CCL exhibits

tetrachloride

protein/min.

different

liver/min.

* Significantly

d nmol/g

’ nmol/rat/min.

b nmol/mg

a Carbon

from control

was administered

2.8

(P
i.p. to groups

of six rats.

+ received

peanut

23.0 20.9

+ 119* 62*

32.9 37.5

213

oil (vehicle).

1.7* + 1.8*

f

+ 1.8 * 2.1

Chloramphenicold

k 134

f

Controls

1536

1939

36.0 f

1000yl/kg 766*

1271

f

11044

49.7

200 $/kg

8 908 +

2362

1149

14406

5’4.2 + 4.8

+ 5.9

2441

+ 1560 f

12415

* 5.0

43.4

Naphthold

Control

Glucuronyltransferase

IN VITRO

Sulfotransferase

II REACTIONS

2-Naphtholb

2-Naphtholc

ON-PHASE

20 &kg

Treatme&

TETRACHLORIDE

& SEM of six rats.

OF CARBON

III

Data are means

EFFECT

TABLE

+ 1.2 + 1.5

Rats were sacrificed

21.0

f 1.6 17.6 + l.O*

24.9

24.0

6.9

k 6.3

+ 7.5

k 7.8*

f

24 h after treatment.

49.1

64.0

90.3

65.1

1,2-Epoxy-3-@-nitrophenoxy)-propaneb

1,2-Dichloro-4nitrobenzeneb

Glutathione-S-transferase

314

TABLE

IV

EFFECT

OF CARBON

Data are means

TETRACHLORIDE

ON PHASE

11 REACTIONS

IN VIVO

& SEM of six rats.

Treatment=

Total

sulfates

Total

excretedbac

glucuronides

Total

excretedbBc

mercapturic

acids excretedb

Control

1.53 & 0.32

(37.8%)

0.86

k 0.11

(21.3%)

16.5 + 1.1

20 /J/kg

2.04

k 0.39

(40.0%)

1.08 & 0.22

(21.2%)

20.6

f

200 /J/kg

1.02 * 0.21

(30.1%)

0.89

+ 0.16

(26.3%)

21.3

+ 1.2

1000 gl/kg

0.54

0.13* (35.1%)

0.39

k 0.06* (25.4%)

20.8

a Carbon

tetrachloride

Rats were sacrificed b Percent

administered

’ Values in parentheses

f

was administered 24 h after dose/24

i.p. to groups

of six rats. Controls

_

2.2

+ 1.9

received peanut

oil (vehicle).

treatment. h.

represent

percent

of chlorophenols

and alcohols

conjugated

as glucuronides

or

sulfates. * Significantly

different

from

control

(P< 0.05).

a biphasic effect on enzyme activity (Tables I and II). The lowest dose induced significantly higher responses both in vitro (Table I) and in vivo (Table II), while the highest dose produced significant inhibition. Though this is the first report of CCL causing significantly higher hepatic mixed function oxidase activity, it has been established that many other chemicals both inhibit and induce hepatic drugmetabolizing enzyme activity depending on the level or treatment schedule used. Thus SKF525A [17], Lilly 18947 [17], Lilly 32391 [17], MG30662 [17], GPA1851 [ 181, piperonyl butoxide [ 191, metyrapone [20], rifampicin [21], bromobenzene [5,22], chlorobenzene [23], methadone [24], propoxyphene [25], and even phenobarbital [26] have been reported to have a biphasic influence on hepatic drug metabolism. The discrepancy

between

the biphasic

influence

of CCL

on phase

I reactions

reported in Tables 1 and 2 and the impaired activity reported in the literature may be due to differences in the administered dose, the substrate employed, the location of the substrate-metabolizing enzyme(s) within the liver lobule [27], and/or the susceptibility of the specific cytochrome P-450 isozymes involved [28,29]. Although CC14 is one of the most widely investigated of all chemicals, there are few studies which have examined the effect of low (< 50 mg/kg) doses on hepatic drug metabolism. There is also some disagreement over the effect of low doses of CCL where they have been investigated. For example, while Kutob and Plaa [30] found increased barbiturate-induced sleeping time from treatment with 0.1 mmol/kg CCL (15.9 mg/kg), Dingell and Heimberg [3 l] determined that 0.1 ml/kg CCL (159.4 mg/kg) produced no significant change in the metabolism of hexobarbital by liver microsomes. However, Dingell and Heimberg also found that 0.1 ml/kg Ccl4 caused a slight but significant decrease in the hepatic metabolism of aminopyrine. Thus, in determining the effect of CCL on hepatic drug metabolism by measuring the rate

315

of metabolism of different substrates in vitro, the choice of substrate may itself be responsible for some of the observed discrepancies in the literature. The distribution of the specific cytochrome P-450 isozymes within the liver lobule, as well as differences in their susceptibility to CC&-induced lesions, can account for such discrepancies in the lack of effect which CCL had on hexobarbital metabolism concomitant with the significant inhibition of aminopyrine metabolism [31]. Such a selectively deleterious effect of CCL on cytochrome P-450-dependent catalytic activities has recently been reported [28,29]. English and Anders [28] found that the isozyme, which catalyzes the metabolism of CC4 to phosgene, was lost at doses of carbon tetrachloride which had little effect on the metabolism of CCL to chloroform. The low dose used in this study, 20 pi/kg CC4 (31.8 mg/kg), lies within a range which was recently reported to produce no apparent toxic effect within a 24 h period [32]. Therefore, it is likely that at low doses of CCL increased enzyme synthesis can still take place, while at higher doses the reduction in protein synthesis combined with the effects of necrosis results in impaired tissue enzyme activity. The effect of CC4 on phase II reactions offered no surprises. Glucuronyl and sulfotransferase were inhibited both in vitro (Table III) and in vivo (Table IV). However, the effect of low levels of CCL on glutathione-S-transferase was less certain. In vivo there was no effect while in vitro conjugation was significantly reduced for only one of the two substrates. Metabolism of the model substrate lindane provided a satisfactory index to the effect of low doses of carbon tetrachloride on the hepatic drug-metabolizing enzymes. REFERENCES Trela,

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