Effects of microsomal enzyme inducers on animals poisoned with hepatotoxins

TOXICOLOGY

AND

APPLIED

PHARMACOLOGY

22,339-346

Effects of Microsomal on Animals Poisoned ARYEH

(1972)

Enzyme Inducers with Hepatotoxinslp2 HURWITZ

Departments of Medicine and Pharmacology, University of Kansas Medical Center, Kansas City, Kansas 66103 Received March 22.1971

Effects of Microsomal Enzyme Inducers on Animals Poisonedwith Hepatotoxins. HURWITZ,A. (1972).Toxicol. Appl. Pharmacol. 22,339-346. The effectsof microsomalenzymeinducerson hepatic function in miceand rats were studied when thesedrugs were given before or after chemical poisoning.If gastricadministrationof phetharbital(50-75 mg/kg/day) was begun 12-18 hr after CCL, (1.4-1.8 ml/kg) was given, the 20 min sulfobromophthalein (BSP) retention returned to normal in 311 days, while animals not receiving the inducer still had markedly abnormal values. SGPT values,which roseafter CCII, werenot affectedby phetharbital. The risein BSPretention after white phosphorus(2-7.5 mg/kg) alsoreturned to normal morerapidly if daily phetharbital followed the administrationof the poison.After phosphorus,SGPT did not riseand phetharbitalhad no effect on the activity of this enzyme. Phenobarbital had the sameeffect as phetharbital on BSP retention if given after phosphorus,but 3,4-benzpyrene, oxyphenbutazoneor cyclizine did not sharethis effect. No inducer affected mortality or weight gain after phosphoruspoisoning of mice. Phenobarbital pretreatment increasedmortality after CCL, but not after phosphorus. No satisfactory treatment is currently available for managementof patients whoselivers have sustained toxic injury. There have been reports that phenobarbital accelerates hepatic excretion of sulfobromophthalein (BSP), one measure of liver function (Klaassen and Plaa, 1968a). Another report states that phenobarbital increases weight,

protein content and metabolic activity of liver regenerating after partial hepatectomy (Frey et al., 1968). Instead of protecting animals from carbon tetrachloride hepatotoxicity, phenobarbital pretreatment renders them more susceptible to the toxic effects of the halogenated hydrocarbons (Garner and McLean, 1969). Therefore, the effects of inducers of microsomal drug oxidation on hepatotoxicity were investigated using carbon tetrachloride, a halogenated hydrocarbon which causes centrilobular necrosis, and white phosphorus, an agent whose effect is primarily on the periportal area of the hepatic lobule (JCdquel, 1958; Ghoshal et al., 1969). ’ Presented in part at the annualmeetingof theFederationof AmericanSocieties for Experimental Biology,Atlantic City, April, 1970. ’ This studywassupportedby Grant GM 15956from the U.S. PublicHealthService. cj 1972 by Academic Press, Inc. 339

340

HURWITZ

METHODS

Male Sprague-Dawley rats and Swiss Webster mice were used in these studies. At the start of each experiment they weighed 1O&l 50 g and 18-22 g, respectively. The animals were fasted overnight before administration of hepatotoxin. Carbon tetrachloride was dissolved in corn oil and given ip. White phosphorus was dissolved in mineral oil and given by gastric intubation. Phetharbital and oxyphenbutazone were suspended in 2 % methyl cellulose in water and given by gastric intubation. Controls received an equal volume of the suspending medium by the same route. Sodium phenobarbital and cyclizine were dissolved in water and 3,4-benzpyrene in corn oil and administered ip. Sodium sulfobromophthalein 125 mg/kg in saline was administered by tail vein, and blood samples were obtained by cardiac puncture 20 min later. BSP serum levels were determined by pipetting a 50 ~1 aliquot into 3 ml of 0.1 N sodium hydroxide and reading in a spectrophotometer at 580 nm. Serum glutamic-pyruvic transaminase (SGPT) activity was determined by the method of Reitman and Frankel(l957). The data were analyzed by the Student t test, Fisher exact test, Chi square test with the Yates correction or Dunnett test where indicated (Sokal and Rohlf, 1969; Dunnett, 1964). RESULTS

When mice are pretreated 4 days with a daily dose of 75 mg/kg sodium phenobarbital before being poisoned with a sublethal dose of carbon tetrachloride, their 24 hr response to carbon tetrachloride as measured by BSP excretion is unaffected (Table 1). By 48 hr, TABLE EFFECT

OF PHENOBARBITAL TETR~HLORIDE

1

PRETREATMENT ON HEPATOTOXICITY AND WHITE PHOSPHORUS IN MICE”

OF CARBON

Treatment Water

Phenobarbital

Pb

24 Hr after poison CC& 0.5 ml/kg CCL 1.5 ml/kg Phosphorus 2 mg/kg Phosphorus 5 mg/kg

37.0 40.0 18.5 22.8

Ccl, 1.O ml/kg Ccl, 1.5 ml/kg Phosphorus 5 mg/kg Phosphorus 7.5 mg/kg

26.2 32.4 36.3 24.7

zk 3.7 zt 2.7 zt 2.1 i 2.9

(8)c (10) (8) (7)

33.2 46.0 12.6 10.6

& k i f

3.1 4.6 0.8 1.1

(8) (4) (8) (8)

NS NS CO.01
56.8 54.3 28.0 29.8

f 4.5 f 6.8 f 6.8 zk 4.8

(4) (4) (4) (5)

<0.005 co.05 NS NS

48 Hr after poison f 3.7 zt 4.7 x!z7.3 * 6.8

(4) (4) (5) (5)

a All animals were given phenobarbital, 75 mg/kg, in water for 4 days. On day 5 they received either carbon tetrachloride in corn oil ip or white phosphorus in mineral oil by gastric tube. Twenty-four or 48 hr later blood was obtained for SGPT activity, and BSP retention (mg/lOO ml) was determined 20 min after an iv dose of 125 mg/kg. Data are means & SE. b Water vs phenobarbital, t test. c Number of mice in each group.

MICROSOMAL

INDUCERS

AND

341

HEPATOTOXICITY

the phenobarbital pretreated animals show greater impairment of hepatic function, retaining more BSP than nonpretreated animals. In contrast, in phosphorus poisoned animals which received phenobarbital, there was more rapid BSP disappearance 24 hr after poisoning. This enhanced hepatic ability to remove BSP was no longer seen 48 hr after poisoning. SGPT, which rises markedly after carbon tetrachloride, remains normal after poisoning with white phosphorus. Phenobarbital pretreatment altered the response to carbon tetrachloride and phosphorus poisoning in yet another way. The lethal dose of phosphorus was unaffected by prior phenobarbital treatment (Fig. 1). Carbon tetrachloride at a dose of 2.25 ml’kg killed 807; of the mice which received phenobarbital, while none of the control animals died after this dose of hepatotoxin. w WATER ‘--*PHENOB$RBITAL :

I'

1.0 1.5 TETRACHLOFUM

2.25

ML/KG

100

75

*LA-.--WHlTE

-.I -_ ~HOSP%3JS

hl%KG

FIG. I. Effect of phenobarbital pretreatment on lethality of carbon tetrachloride and white phosphorus in mice. All animals were given phenobarbital 75 mg/kg ip for 4 days. On day 5 they received carbon tetrachloride in corn oil ip or white phosphorus in mineral oil by gastric tube. The number of mice dying within 48 hr was then determined. Those who survived the first 48 hr recovered from the poison. Each point represents the data from a group of 7 or 8 mice. *P < 0.01, chi-square test.

Since both carbon tetrachloride and phenobarbital cause central nervous system depression, phetharbital, a type I inducer with less CNS depressant properties (Kuntzman et al., 1968), was given after carbon tetrachloride. As seen in Table 2, by day 3 after TABLE 2 EFFECTOF PHETHARBITALON RECOVERYFROM CARBONTETRACHLORIDE INTOXICATION IN RATS"

Treatment Methylcellulose Corn oil CC& PC

58.5 z?z1.8 (4)d 71.4 i 4.3 (7) co.05

Phetharbital

.--~~~ .45.4* 2.2 (3) 47.3 -f 1.5 (7) NS

Ph -co.01 CO.001

a Carbon tetrachloride in corn oil was administered ip at 1.2 ml/kg. Six, 24 and 48 hr later the rats received phetharbital in 2% methyl cellulose, 50 mg/kg, or methyl cellulose by gastric tube. One day after the third gastric intubation, BSP (125 mg/kg) was given by tail vein. BSP retention (mg/lOO ml) was determined 20 min later. Data are means f SE. b Methyl cellulose vs phetharbital, t test. ’ Corn oil vs carbon tetrachloride, t test. d Number of rats in each group.

342

HURWITZ

poisoning those animals receiving phetharbital had normal BSP disappearance compared to controls. Furthermore, as expected (Klaassen and Plaa, 1968a), unpoisoned rats which were given phetharbital also exhibited less BSP retention than controls. The time course of this recovery of hepatic function as reflected by BSP and SGPT is seen in Fig. 2. Those animals which received phetharbital after carbon tetrachloride poisoning had a significantly improved ability to handle BSP in 3-4 days when they were compared with methyl cellulose controls. The SGPT activities, which were also returning to normal, showed no difference in the 2 groups. u METHYLCELUOSE - - 4 MTHARBITAL

!

I

2 DAYS

3

150 I

4

2

OAYS

3

4

FIG. 2. Effect of phetharbital on recovery from carbon tetrachloride intoxication in mice. All animals were given carbon tetrachloride 1.8 ml/kg in corn oil ip. They were started 16 hr later on daily gastric doses of 2 % methyl cellulose either with or without phetharbital, 50 mg/kg. Daily SGPT activities and BSP retentions 20 min after 125 mg/kg iv were determined. Controls received no carbon tetrachloride. Their BSP retentions were determined after 4 daily doses of phetharbital or methyl cellulose. Each point represents the mean f SE determined from a group of 6-8 mice. *P -c0.05,t test.

+--

2

DAYS

3”

4

FIG. 3. Effect of phetharbital on recovery from white phosphorus intoxication in mice. The mice were given white phosphorus in mineral oil 5 mg/kg or mineral oil by gastric tube. After 6 hr they were started on daily gastric doses of 2 % methylcellulose, with or without phetharbital, 50 mg/kg. Daily SGPT activities and BSP retentions 20 min after 125 mg/kg iv were determined. Controls received no phosphorus. Their BSP retentions were determined after 4 daily doses of phetharbital or methylcellulose. Each point represents the mean f SE determined from a group of 68 mice. *Pi 0.05,t test.

The accelerated return to normal BSP clearance due to phetharbital was also seen after poisoning with white phosphorus, whose effect is more prolonged (Fig. 3). By day 4 the BSP clearance of phosphorus poisoned mice receiving phetharbital was within the normal range, while those which were given methyl cellulose still had markedly

MICROSOMAL

INDUCERS

AND

TABLE EFFECT OF MICROSOMAL F~KTION IN MICE

343

HEPATOTOXICITY

3

ENZYMEINDUCERS ON RECOVERY OF HEPATIC AFTER WHITE PHOSPHORUS INTOXICATION” 20-Min BSP retention (mg/lOO ml)

Mineral oil-water (9) Phosphorus-Water(13) -3,4-Benzpyrene20 mg/kg/day (I 3) -Cyclizine 25 mg/kg/day (8) -0xyphenbutazone 50 mg/kg/day (9) -Phenobarbital 50 mg/kg/day (12) -Phetharbital 50mg/kg/day (9)

Pb

15.8f: 3.4 29.1 s. 3.3 33.1:t 3.4 32.2 + 4.1 31.1 3: 5.0 20.1 + 3.1 18.5+ 2.8

NS NS NS co.05 -10.05

’ After an overnightfast, on day 1 miceweregivenmineraloil with or without whitephosphorus, 7.5mg/kgby gastrictube;6 hr later,andondays2,3 and4 themiceweredosedwith theinducingagents. On day 5 BSPretentions(20min after 125mg/kgiv) weredetermined.Data are meansf SE. bP valuescompareBSPretentionsin phosphorus poisonedmicewhichweregivenwaterwith those whichreceived inducers, t test. c Numberof micein eachgroup. abnormal BSP retention. SGPT activities, which appeared to be rising gradually after phosphorus poisoning, remained within the normal range and were not affected by phetharbital treatment. Since phetharbital acceleratesrecovery of hepatic excretory function, several additional agents, all of which stimulate microsomal enzyme induction, were evaluated to determine whether they restore BSP removal to normal in mice after white phosphorus poisoning (Table 3). An antihistamine, cyclizine, was tried, since Rees(1965) reported TABLE EFFECT

OF MICROSOMAL

AND SURVIVAL

IN MICE

ENZYME

4 INDUCERS

AFTER WHITE

ON BODY

PHOSPHORUS

WEIGHT

POISONING’

Dead __~ “/;,.-~ Day 3 Day 4 Mineral Oil-Water Phosphorus-Water -3,4-Benzpyrene20 mg/kg/day -Cyclizine 25 mg/kg/day -0xyphenbutazone 50mg/kg/day -Phenobarbital 50 mg/kg/day -Phetharbital50 mg/kg/day

0 (O/15)” 35 (6/17)* 50 (6/l 2)* 50 (6/12)*

38 (S/16)* 50 (s/16)* 56 (9/16)*

Body weight (g) Day 4

13 (2/15)b 59 (10/17)*

26.2 it 0.5

58 58 44 50 62

17.0 18.0 17.1 17.5 17.3

(7j12)* (7/12)* (7/16) (8/16) (10/16)*

16.5 .k 0.6* f 0.5*

+ l.O* IL 0.4* t0.8* t 0.4

aAfter an overnightfast, on day 1 miceweregivenmineraloil with or without whitephosphorus, 6 mg/kgby gastrictube.Sixhr latertheyreceived+ the indicateddoses of inducingagents.Ondays2,3 and4 theyreceivedfull dosesof inducingagents,andthe numberof survivorsandtheir weightswere recorded. * Numberdeadin eachexperimentalgroup. * P r_0.05by Fisherexacttest for mortality andDunnett tablefor weights.

344

HURWITZ

that this group of drugs protects against carbon tetrachloride toxicity. Oxyphenbutazone has been shown to lower transaminase activities in marmosets infected with hepatitis virus (Mustala, 0. O., personal communication). 3,CBenzpyrene, which is ineffective in stimulating BSP excretion in rats (Klaassen, 1970), is a type II inducer related structurally to pyrene which stimulates 3-methyl, 4-methyl-aminobenzene metabolism in mice (Brown et al., 1954). Phenobarbital and phetharbital were the only drugs studied which consistently accelerated BSP removal in mice poisoned with phosphorus. Despite accelerated recovery of “hepatic function” as reflected in BSP removal, all the animals poisoned did poorly and appeared grossly sick. Table 4 shows that whether or not any inducer was given after phosphorus poisoning, the animals failed to gain weight and no drug caused increased survival. DISCUSSION Previous studies have explored the interactions of microsomal enzyme inducers with hepatotoxins and hepatic regeneration. Some of these have suggested that inducers increased damage while others implied that they may be beneficial. Carbon tetrachloride is known to cause centrilobular necrosis of the liver, with impaired protein synthesis (Richter et al., 1968; Monlux and Smuckler, 1969), damaged endoplasmic reticulum (Meldolesi et al., 1968), lowered P-450 levels (Sasame et al., 1968 ; Barker et al., 1969) and impaired hepatic excretory function as measured by BSP retention (Klaassen and Plaa, 1968b). Phenobarbital brings about the reversal of many of these manifestations of toxicity, stimulating an increase in growth (Argyris, 1968), protein synthesis (Frey et al., 1968), drug metabolism (Argyris, 1968), P-450 synthesis (Gram et al., 1968) and more rapid regeneration and restoration of function after partial hepatectomy (Chiesara et al., 1967; Argyris, 1968; Frey et al., 1968). Klaassen and Plaa (1968a) have shown that phenobarbital accelerates BSP excretion and bile flow. Despite these apparently beneficial effects, phenobarbital pretreatment has been shown to increase carbon tetrachloride toxicity, supposedly by stimulating conversion of carbon tetrachloride to a toxic metabolite (Garner and McLean, 1969). These observations showing that the mortality of carbon tetrachloride was markedly increased by phenobarbital pretreatment were confirmed in the present study. White phosphorus, like carbon tetrachloride, has been reported to cause lipoperoxidation (Ghoshal et al., 1969). Pretreatment with phenobarbital had no effect on phosphorus lethality, unlike the effect on carbon tetrachloride. The effects of phenobarbital pretreatment on the excretory function of the chemically damaged liver also varied depending on the nature of the poison. BSP excretion in phenobarbital pretreated animals was unaffected 24 hr after carbon tetrachloride but was depressed by 48 hr. BSP excretion in animals given phenobarbital before white phosphorus poisoning is greater 24 hr after the phosphorus than in animals not induced, but this effect disappears within the next 24 hr. Using BSP excretion as a measure of hepatic function and SGPT as an indication of hepatocellular damage, the effects of treatment with microsomal enzyme inducers on hepatic recovery after exposure to poison were investigated. In place of phenobarbital, phetharbital was used in these experiments because of the difference in CNS depressant properties of these inducers. In contrast to the effect of inducer pretreatment, if phetharbital was given after poisoning, BSP excretory capacity returned to normal after

MICROSOMAL INDUCERS AND HEPATOTOXICITY

345

damage with either carbon tetrachloride or phosphorus. Phetharbital had no effect on the SGPT response to either poison, neither to the marked rise after carbon tetrachloride nor to the activity after phosphorus which did not become abnormal. In conclusion, it appears that pretreatment with microsomal enzyme inducer affects differently the hepatotoxicity of the centrilobular poison, carbon tetrachloride, and white phosphorus, which causes periportal damage. After poisoning with either of these agents, some inducers, including phenobarbital and phetharbital, accelerate the recovery of the hepatic ability to remove BSP from the plasma. This ability to remove BSP does not correlate with increased survival or weight gain. Therefore, the usefulness of these drugs in the treatment of toxic liver injury is doubtful. Furthermore, the BSP test may not be a valid measure of some aspects of hepatic function necessary for survival. Although the SGPT activities rose markedly after carbon tetrachloride, they were a very poor indicator of exposure to sublethal doses of white phosphorus which caused sustained elevation of BSP. ACKNOWLEDGMENT I thank Dr. K. Hassaneinfor his suggestionson the statisticalevaluation of the data.

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