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Enhanced activity of liver drug-metabolizing enzymes for aromatic and chlorinated hydrocarbons following food deprivation
TOXICOLOGY
AND
APPLIED
PHARMACOLOGY
50,549-556
(1979)
Enhanced Activity of Liver Drug-Metabolizing Enzymes for Aromatic and Chlorinated Hydrocarbons following Food Deprivation T. NAKAJIMA AND A. SATO Department
of Hygiene, School of Medicine,
Shinshu University, 390 Japan
Asahi, Matsumoto,
Received January 16, 1979; accepted May 16, 1979
EnhancedActivity of Liver Drug-MetabolizingEnzymesfor Aromatic andChlorinated HydrocarbonsfollowingFood Deprivation.NAKAIIMA, T., AND SATO, A. (1979).Toxicol. Appl. Pharmacol. 50, 549-556. The in vitro activity of liver microsomal enzymesto metabblize somearomaticand chlorinatedhydrocarbonswasenhancedremarkablyin l-day fastedratsof bothsexes, althoughfastingproducednosignificantincrease in themicrosomal protein and cytochromeP-450 contents.Eventhe activity per wholeliver wasat a significantly increased level, despitethe fact that fastingcauseda markedlossof liver weight. A sexdifferencewasnotedin the metabolism of thesehydrocarbonsboth in fed and l-day fastedrats. However,food deprivationfor 3 dayswasfound to decrease the extentof this sex difference.A metabolismstudy usingtrichloroethyleneasa modelsubstrateshowed that the rate of metabolism in vivo wasalsoenhanced by fasting.
Food deprivation has been reported to produce a variety of substrate- and sex-related effects on liver drug-metabolizing enzymes in rats: After 72 hr it decreased aminopyrine and hexobarbital metabolism in male rats but increased them in female rats, whereas it increased the activity of aniline hydroxylase in both sexes (Kato and Gillette, 1965). It is also known that food deprivation potentiates the hepatotoxicity of some chlorinated hydrocarbons such as carbon tetrachloride (Krishnan and Stenger, 1966; Diaz G6mez et al., 1975; Jaeger et al., 1975), chloroform (Davis and Whipple, 1919; Goldschmidt et al., 1939), and l,l-dichloroethylene (Jaeger et al., 1973c, 1974, 1975; McKenna et al., 1978). However, very little has hitherto been known regarding the effects of food deprivation on the metabolism of these hydrocarbons.
We have developed recently a very simple method for evaluating the liver drug-metabolizing enzyme activity using a gas chromatographic technique (Sato and Nakajima, 1979), which can be applied to enzyme studies in vitro of almost all of the volatile hydrocarbons. In the present report, the effects of food deprivation on the in vitro and in vivo metabolism in rats of both sexesof some aromatic and chlorinated aliphatic hydrocarbons have been described. METHODS Animals. Wistar male and femalerats were fed
pellet food (Nippon Clea, Type E-2) and water ad Iibitum until they reacheda weight of 250g in
maleand 150g in femalerats.Onreachingthe weight, they were fed a nutritionally adequateliquid diet (1Cal/ml) as previously reported (DeCarli and Lieber, 1967).The diet was given daily at 4 PM,
549 All
0041-008X/79/12054948$02.00/0 Copyright Q 1979 by Academic Pres, Inc. rights of reproduction in any form reserved. Printed in Great Britain
550
NAKAJIMA
AND
80 ml to male and 70 ml to female rats, and rats were usually sacrificed at 10 AM for enzyme assay. The feeding was continued for at least 3 weeks before sacrifice. Food deprivation was caused by substituting an equivolume of tap water for the liquid diet. The period of time between feeding and sacrifice was an important factor affecting the activity of liver drug-metabolizing enzymes (unpublished observations). In this regard, the liquid diet feeding had an advantage in our metabolism study over the normal pellet food feeding; rats drank the liquid diet given at 4 PM almost completely by 10 PM on the same day. Chemicals. Nicotinamide-adenine dinucleotide phosphate disodium salt (NADP) and glucose-6phosphate disodium salt (G-6-P) were obtained from Boehringer-Mannheim, and the other chemicals used were of reagent grade purchased from Nakarai Chemicals, Kyoto. Metabolism study in vitro. Rats were decapitated followed by exsanguination. The liver was minced and a 10% (w/v) crude homogenate was made with 1.15 % KCI-O.01 M Na+/K+-phosphate buffer, pH 7.4, in a glass homogenizer. The homogenate was then centrifuged at 30,OOOg for 10min. A part of the resultant supernatant was further centrifuged at 105,OOOg for 60 min to obtain microsomal pellets. One milliliter of the 10,000g supernatant was normally used as the source of the enzyme. One-tenth of a milliliter of a water solution of hydrocarbons, mostly 5 to 10 ~1/100 ml, was used as the substrate. The enzyme and substrate were incubated with 2 ml cofactor solution (containing: 0.5 prnol NADP; 10 pmol G-6-P; 25 ,umol MgCI,) in an airtight vessel for 10 min. The rate of metabolic rection was assessed by measuring the rate of substrate disappearance and was generally expressed as nanomoles per gram of liver per minute (Sato and Nakajima, 1979).
Microsomal
protein
Fed l-Day fast 2-Day fast 3-Day fast
334.9 * 17.36 320.2k 10.0 298.8 k 8.0 297.5 f 9.5
Liver (9)
P-450
contents.
RESULTS EfSect of Food Deprivation on Body Weight, Liver Weight, and Microsomal Protein and Cytochrome P-450 Contents
After l-day fasting, male rats lost 5% and female rats 4% of their original body weights. Male rats had about 25% and female rats 20% less liver weight than the respective 1
ON BODY AND LIVER WEIGHTS
Male Body (g)
and cytochrome
The microsomal protein content was measured according to Lowry et al. (1951) as modified by Miller (1959). The amount of cytochrome P-450 was estimated by the method of Omura and Sato (1964). Effect of food deprivation on trichIoroethylene metabolism in vivo. In the in vivo metabolism study, trichloroethylene was selected as a model substrate, because the metabolites excreted in urine are not found normally in urine, thus urine analysis gives more accurate information about the effect of food deprivation. Four fed and four l-day fasted, male rats were exposed to 500 ppm of trichloroethylene for 4 hr (8 to 12 AM) in a dynamic airflow exposure chamber as previously reported (Sato et al., 1975a). After cessation of exposure the concentration of trichloroethylene in blood and the amount of its urinary metabolites (total trichloro compounds, TTC) were measured at varying times. Trichloroethylene concentration in blood was measured by a syringe-equilibration method (Sato et al., 1975b). Urinary TTC was estimated as described by Tanaka and Ikeda (1968). Statistical analysis. Results were compared by analysis of variance (Table 1, Fig. 1) or Student's t test (Tables 2 and 3).
TABLE EFFECTS OF FOOD DEPRIVATION
SAT0
Female Liver/body”
Body (8)
Liver 64 1.8
Liver/body”
9.74kO.73
2.9OkO.20
182.3+
6.2OkO.28
3.40&0.15
7.58kO.38
2.39kO.11'
178.7k8.8
5.08kO.23
2.84+0.02c
6.7OkO.26
2.24_+0.09c*d
162.5k4.8
4.25kO.37
2.61&0.07+'
6.17kO.06
2.07~0.08c~d~e
167.8k9.3
3.97kO.40
2.36~0.11c~d~e
0 Liver weight per 100 g body wt. b The values represent the mean f SD for five rats. c Significantly different from fed rats, p < 0.05. d Significantly different from l-day fasted rats, p x 0.05. e Significantly different from 2-day fasted rats, p < 0.05.
FASTING
ON HYDROCARBON
TABLE
551
METABOLISM
2
EFFECTSOF Foot DEPRIVATION ON LIVER MICROKIMAL PROTEIN AND CYT~CHROME P-450 C0m~Nm Male Animals
Protein (mg/g liver)
Fed l-Day fast 2-Day fast 3-Day fast
22.7 f 2.7” 23.0+ 2.7 22.0 + 2.0 22.3k2.4
Female
P-450 (nmol/mg protein)
Protein (mg/g liver)
0.842*0.123* 0.823 &-0.030b 0.782 f 0.042b 0.799 + 0.090*
22.5+ 23.7+ 23.6 + 22.1+
P-450 (nmol/mg protein)
1.5 1.7 2.4 0.7
0.638kO.051 0.673 + 0.044 0.633 i- 0.039 0.638 + 0.023
a The values represent the mean+ SD for five rats. b Significantly different from the respective female rats, p < 0.05. TABLE
3
EFFECT OF I-DAY Foot DEPRIVATION ON THE ACTIVITY OF DRUG-METABOLIZING ENZYMES” Metabolic rate (nmol/g/min) Male Substrates Benzeneb*c*d Tolueneb~c*d~e m-Xyleneb*c,d Ethylbenzeneb~c*d*e n-Propylbenzeneb*‘*@ Cumeneb*Csd@ Styreneb*C*d*e Dichloromethaneb*c*d*e Chloroform**‘** Carbon tetrachlorideb*c*dse 1, l-Dichloroethanec~d~’ 1,2-Dichloroethaneb*csd*e l,l,l-Trichloroethane’ 1,l ,2-Trichloroethaneb*c*d*e 1,1,1,2-Tetrachloroethanec*d 1,1,2,2-Tetrachloroethanebvcsd Trichloroethyleneb*‘sd Tetrachloroethylene’ 1, l-Dichloroethyleneb*c*d l-Chloropropaneb~c~d*e Monochlorobenzenec*d
Fed 13.7* 5.4 18.lk4.9 21.0* 5.5 22.9k6.3 46.5 + 12.8 36.6k4.3 28.5k4.3 28.5r1.5 19.7k2.6 1.9kO.2 19.lk3.3 23.6+ 1.1 0.5 + 0.2 21.Ok1.9 8.1 k2.0 13.3kO.6 18.9+ 7.4 0.5 f 0.3 31.1 k6.6 37.6+ 5.9 8.2? 1.6
Fasted 36.4+ 2.8 40.3 + 4.8 41.0* 1.9 40.8 + 3.5 77.2+ 17.4 75.9k 1.6 46.1+ 1.6 60.9+ 1.9 55.1 + 7.5 5.9kO.8 56.0& 2.4 59.8 + 3.1 1.2kO.2 56.0+ 3.0 32.8 + 8.4 40.0 f 2.4 57.0+ 1.9 1.950.3 67.3 k 6.6 lOO.Ok 5.2 35.5 f 6.3
Female Fasted/fed
Fed
2.7 2.2 2.0 1.8 1.7 2.1 1.6 2.1 2.8 3.1 2.9 2.5 2.4 2.7 4.0
8.5 + 0.2 10.6k4.1 16.3 f 3.6 13.5k4.5 24.7 It 5.9 14.6f 4.5 16.8 I? 3.2 21.5k5.5 15.3k6.8 1.1 kO.5 14.6+ 1.6 14.8rt4.1 0.4kO.3 12.9+ 1.3 6.9k2.7
29.3k1.2 29.9k1.6 33.3 + 3.5 27.8 + 1.8 50.8 -+ 3.9 26.9 + 3.5 36.1+ 6.5 51.7k2.9 39.3k2.5 4.520.3 48.5+ 12.2 53.3k2.9 0.8kO.5 46.8 k 8.3 26.9_+ 6.7
3.4 2.8 2.0 2.1 2.1 1.8 2.1 2.4 2.6 4.1 3.3 3.6 2.0 3.6 3.9
3.0 3.0 3.8 2.2 2.7 4.3
15.725.2 13.4k3.8 0.7kO.5 25.4k2.7 17.8k2.9 lO.O& 0.8
30.5k1.6 38.4k2.4 1.8+ 1.5 44.6+ 4.2 40.8 f 10.2 31.3k5.6
1.9 2.9 2.6 1.8 2.3 3.1
a The values in the columns Fed and Fasted represent the meanf SD for five rats. * Significantly different between male and female fasted rats, p -c0.05. c Significantly different between fed and fasted male rats, p <: 0.01. d Significantly different between fed and fasted female rats, p-c 0.01. e Significantly different between male and female fed rats, p -c 0.05.
Fasted
Fasted/fed
552
NAKAJIMA AND SAT0
control fed rats. When food deprivation was the metabolic rates of all the hydrocarbons continued over a period of 2 or 3 days, the studied were markedly elevated in animals of body and liver weights were further de- both sexes. With all the marked loss of liver creased. Although the loss during this period weight in fasted rats, the total liver enzyme was somewhat less conspicuous than the activity (activity/whole liver/unit time) in loss caused by l-day fasting, a significant de- these rats was still significantly higher than crease in liver weight per 100 g body wt was that in fed rats. noted with this prolongation of food deprivation. These findings indicate that the liver is Sex Deference in the Effect of Food Deprivation on Drug-Metabolizing Enzyme Activity more seriously affected by food deprivation than the total body weight would suggest As shown in Table 3, male fed rats meta(Table 1). bolized most of the hydrocarbons more On the other hand, food deprivation pro- rapidly than fed female rats. This sex differduced no obvious effect on the microsomal ence in enzyme activity became much more protein and cytochrome P-450 contents noticeable after 1 day of fasting. (Table 2). However, when food deprivation was conSex differences were found in body weight, tinued over a period of 2 or 3 days, no further liver weight, and cytochrome P-450 content, significant increase of the enzyme activity but not in microsomal protein content. occurred in male rats, whereas the activity in female rats tended to further increase Activation of Liver Drug-Metabolizing En- during this prolonged fast (Fig. 1). Consezymes by Food Deprivation quently, the sex difference in the enzyme Metabolic rates of hydrocarbons in vitro in activity, which was evidently observed both fed and fasted rats are listed in Table 3. in the well-fed and l-day fasted rats, became Clearly, the liver of rats of both sexes meta- less apparent with the prolongation of fasting. bolized 1,l , I-trichloroethane, tetrachloroAs the liver weight decreased with proethylene, and carbon tetrachloride much less longed food deprivation, the enzyme activity rapidly than the other hydrocarbons. per whole liver in rats of both sexes was When the liquid diet was replaced by an lowered during this term compared with the equivolume of water 1 day before sacrifice activity in l-day fasted rats. 70-
-*-MALE -.a.--FEMALE
8 00. ? $ 50e
TOLUENE
i
11
BENZENE
0-e
0
12
3
olza DURATION
OF
FAST.
DAYS
FIG. 1. Activity of liver drug-metabolizing enzymes in relation to duration of food deprivation. Each point represents the meanf SD for five rats. (*) Significantly different from l-day fasted female rats, p < 0.05.
FASTING
ON HYDROCARBON
553
METABOLISM
these reconstituted 10,000g supernatants. As is shown in Table 4, the enzyme activation following food deprivation is associated with changes in the microsomes, but not in the Since the 10,000g supematant was used as soluble fraction. the enzyme source, there arises a question of Efect of Food Deprivation on Apparent K, whether the enhanced activity by starvation and V,,,,, for Toluene and Trichloroethylene has been derived from changes in microsome Metabolism or soluble fraction. The 10,000g supematant was centrifuged at 105,OOOg for 1 hr and Figure 2 shows the double reciprocal plots divided into the soluble and microsomal of metabolic rates of toluene and trichlorofractions. The soluble fraction from a fed ethylene versus substrate concentrations. rat was added to the microsome from a Clearly, not only the maximum velocity (V,,J but also the Michaelis constant (K,) fasted rat, and conversely the soluble fraction from the fasted rat to the microsome from changed with food deprivation. The values the fed rat. The metabolic rates of toluene for K, and V,,, obtained from Fig. 2 were and trichloroethylene were measured with as follows, respectively: 3.3 PM and 16.7
Metabolic Rates of Toluene and Trichloroethylene Measured by Interchanging Microsome and Soluble Fraction between Fed and Fasted Rats
TABLE
4
METABOLIC RATES OF TOLUENE AND TRICHLOROETHYLENE INTERCHANGING MICR~SOME (M) AND SOLUBLE FRACTION FED AND FASTED RATS=
MEASURED BY (S) BETWEEN
Metabolic rate (nmol/g/min) Enzyme sources (M) (M) (M) (M)
from from from from
Toluene
fed rat+(S) from fed rat fed rat+(S) from fasted rat fasted rat + (S) from fasted rat fasted rat+(S) from fed rat
Trichloroethylene
17.4k2.2 17.2+ 1.8 42.2+ 3.3 4O.lk2.1
18.1k1.3 18.0+ 1.2 56.0+ 2.7 52.4k2.9
a The values represent the mean f SD of five measurements. -.-FED --*--
7 0.2-
FASTED
TOLUENE
s z i ,G 0.1.
0.2-
‘:
?‘ii
TRICHIDROETHYLENE
3 e /’
f 0,’
,
_/‘. /
p.,’
,’
,x3-.
l/s. ,uM-’
,<
O.l-
A’
/p
,A’
,.o. /D.--,’
‘/S.
AIM-’
FW 2. Double reciprocal plots of metabolic rate (u) versus substrate concentration represents the mean for three fed or three l-day fasted male rats. .
(s). Each point
554
NAKAJIMA
AND
nmol/g/min in fed rats and 12.5 ,UM and 41.7 nmol/g/min in fasted rats for toluene; 3.8 PM and 20.0 nmol/g/min in the fed rats and 16.7 ,UM and 55.6 nmol/g/min in the fasted rats for trichloroethylene. Effect of Food Deprivation on the Transfer of Trichloroethylene in Male Rats
The time course of trichloroethylene concentration in blood and that of cumulative urinary excretion of its metabolites (TTC) are shown in Figs. 3 and 4, respectively. The postexposure concentration of trichloroethylene in blood was significantly lower in fasted rats than in fed rats. On the other hand, the fasted rats excreted a much greater amount of TTC in 24 hr after start of exposure than the fed rats. Particularly, the amount of TTC excreted during the first 5 hr was much larger in fasted rats than in fed rats. DISCUSSION A l-day fast decreased the liver weight of rats by about 20% without significant changes in microsomal protein and cytochrome P-450 contents. However, the fasting caused a remarkable increase in the activity of liver microsomal drug-metabolizing enzymes; even the activity per whole liver was at a
SAT0
significantly increased level in spite of the marked loss of liver weight in the fasted rats (Tables 1 and 2). This activation of enzymes following fasting was certainly associated with some changes in the microsomal fraction but not related to a change in the concentration of microsomal protein or cytochrome P-450. These findings suggest that the enzyme activation by fasting may differ from the enzyme
0.01 1. 0
a 1
a 2
4
6
8
HOURS
FIG. 3. Time courses of trichloroethylene concentration in blood after 4-hr exposure to 500 ppm of trichloroethylene. Each point represents the mean + SD for four fed or four l-day fasted male rats.
POST - EXPOSURE
Ok+4 0
t 45
5
8
10
12
14
24
HOURS
FIG. 4. Cumulative amount of TTC excreted in urine during and after 4-hr exposure to 500 ppm of trichloroethylene. Each point represents the mean f SD for four fed or four l-day fasted male rats.
FASTING ON HYDROCARBON METABOLISM
induction of phenobarbital which causes an increase of microsomal protein and cytochrome P-450 content (Conney, 1967). However, the underlying mechanism of the enzyme activation is not yet clear. In the in uivo metabolism study with trichloroethylene, its concentration in blood following inhalation exposure was found to be much lower in fasted rats than in fed rats, hence suggesting that its concentration in liver would have been lower in the former. Nevertheless, the fasted rats excreted about 50% as much urinary TTC than did the fed rats. Therefore, it can be said with certainty that food deprivation increases the metabolism of trichloroethylene not only in vitro but also in uiuo. Food deprivation has been reported to affect the activity of liver drug-metabolizing enzymes in a sex-related manner: it decreases the metabolism of aminopyrine in male rats but increases in female rats, whereas it increases the metabolism of aniline in both sexes (Kato and Gillette, 1965). Although an apparent sex difference was found in the activity of enzymes to metabolize most of the hydrocarbons studied, food deprivation activated the enzymes almost to an equal extent in both sexes. Some chlorinated hydrocarbons have been known to cause more serious liver damage in fasted rats than in fed rats (Davis and Whipple, 1919; Krishnan and Stenger, 1966; Jaeger et al., 1973c, 1974, 1975; Diaz Gomez et al., 1975; McKenna et al., 1978). However, the biochemical mechanism for this enhanced susceptibility of fasted rats has not been fully elucidated. The hepatic necrosis due to carbon tetrachloride is caused not by the hydrocarbon itself but by its active metabolites produced in the liver (Butler, 1961; Slater, 1966; Recknagel and Glende, 1973). In our present investigation, l-day fasting of rats increased the in vitro metabolism of carbon tetrachloride by about three to four times, suggesting that fasting may lead to a greater production of the toxic intermediates and
555
hence to a more severe liver damage. This may offer a possible explanation for the increased carbon tetrachloride-induced hepatotoxicity observed in fasted rats. Brief or 1%hr fasting has also been known to increase the susceptibility of rats to the hepatotoxicity of chloroform (Davis and Whipple, 19 19) and 1,l -dichloroethylene (Jaeger et al., 1973c, 1974, 1975; McKenna et al., 1978). In this case, the decreased concentration of hepatic glutathione (GSH) is said to be responsible for the increased susceptibility in the fasted rats, since GSH is greatly involved in the detoxification pathway for these hydrocarbons (Jaeger et al., 1973a, 1974; Docks and Krishna, 1976; McKenna et al., 1978). It is also generally accepted that chloroform (Ilett et al., 1973) and l,l-dichloroethylene (Jaeger er al., 1974; Bartsch et al., 1975) are activated to respective proximate toxicants, although a possibility that 1,I-dichloroethylene itself causes hepatic injury cannot be denied (Carlson and Fuller, 1972; Jenkins ef al., 1972; Jaeger et al., 1973b). In our metabolism study in vitro, the metabolism of chloroform and l,l-dichloroethylene in the liver of food deprived rats increased about three-fold. Therefore, it can be proposed that the elevated metabolism following food deprivation may also play an important role in increasing the susceptibility of fasted rats to these hepatotoxic hydrocarbons. REFERENCES BARTSCH, H., MALAVEILLE, C., MONTESANO, R., AND TOMATIS, L. (1975). Tissue-mediated mutagenicity of vinylidene chloride and 2-chlorobutadiene in Salmonella typhimurium. Nutwe (London) 255, 641-643. BUTLER,
T. C. (1961). Reduction of carbon tetrachloride in uico and reduction of carbon tetrachloride and chloroform in vitro by tissues and tissue homogenates. J. Pharmacol. Exp. Ther. 134, 311-319. CARLSON, G. P., AND FULLER, G. C. (1972). Interaction of modifiers of hepatic microsomal drug metabolism and the inhalation toxicity of l,ldichloroethylene. Rex Commun. Chem. Pathol. Pharmacol.
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556
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A. H. (1967). Pharmacological implication of microsomal enzyme induction. Pharmacol. Rev.
CONNEY,
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DAVIS, N., AND WHIPPLE, C. (1919). The influence of fasting and various diets on the liver injury effected by chloroform anesthesia. Arch. Znt. Med. 23, 612-633. DECARLI, L. M., AND LIEBER, C. S. (1967). Fatty liver in the rat after prolonged intake of ethanol with a nutritionally adequate liquid diet. J. Nutr. 91, 331-336. DfAz G~MEZ, M. I., DE CASTRO, C. R., DE FERREYRA, E. C., D’ACOSTA, N., DE FENOS, 0. M., AND CASTRO, J. A. (1975). Mechanistic studies on carbon tetrachloride hepatotoxicity in fasted and fed rats. Toxicol. Appl. Pharmacol. 32, 101-108. DOCKS, E. L., AND KRISHNA, G. (1976). The role of glutathione in chloroform-induced hepatotoxicity. Exp. Mol. Pathol. 24, 13-22. GOLDSCHMIDT,
S., VARS,
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RAVDIN,
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(1939). The influence of the foodstuffs upon the susceptibility of the liver to injury by chloroform, and the probable mechanism of their action. J. Clin. Invest. 18, 277-289. ILETT, K. F., REID, W. D., SIPES,I. G., AND KRISHNA, G. (1973). Chloroform toxicity in mice: Correlation of renal and hepatic necrosis with covalent binding of metabolites to tissue macromolecules. Exp. Mol. Pathol. 19, 215-229. JAEGER, R. J., CONOLLY, R. B., AND MURPHY, S. D. (1973a). Diurnal variation of hepatic glutathione concentration and its correlation with l,l-dichloroethylene inhalation toxicity in rats. Res. Commun. Chem. Pathol. Pharmacol. 6, 465471. JAEGER, R. J., TRABULUS, M. J., AND MURPHY, S. D. (1973b). Biochemical effects of l,l-dichloroethylene in rats: Dissociation of its hepatotoxicity from a lipoperoxidative mechanism. Toxicol. Appl. Pharmacol. 24, 457-467. JAEGER, R. J., CONOLLY, R. B., AND MURPHY, S. D. (1974). Effect of 18 hr fast and glutathione depletion on l,l-dichloroethylene-induced hepatotoxicity and lethality in rats. Exp. Mol. Pathol. 20, 187-198. JAEGER,
R. J., CONOLLY,
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(1975). Short-term inhalation toxicity of halogenated hydrocarbons. Effects on fasting rats. Arch. Environ. Health 30, 26-30. JAEGER, R. J., TRABULUS, M. J., AND MURPHY, S. D. (1973~). The interaction of adrenalectomy, partial adrenal replacement therapy, and starvation with hepatotoxicity and lethality after l,l-dichloro-
ethylene intoxication. Toxicol. Appl. Pharmacol. 25, 491. JENKINS, L. J., TRABULUS, M. J., AND MURPHY, S. D. (1972). Biochemical effects of l,l-dichloroethylene in rats: Comparison with carbon tetrachloride and 1,2-dichloroethylene. Toxicol. Appl. Pharmacol. 23, 501-510. KATO, R., AND GILLETTE, J. R. (1965). Effect of starvation on NADPH-dependent enzymes in liver microsomes of male and female rats. J. Pharmacol. Exp. Ther. 150, 279-284. KRISHNAN, N., AND STENGER, R. (1966). Effects of starvation on the hepatotoxicity of carbon tetrachloride. Amer. J. Pathoi. 49, 239-246. LOWRY, 0. H., ROSEBROUGH, N. J., FARR, A. L., AND RANDALL, R. J.,(1951). Protein measurement with the Folin phenol reagent. J. Biol. Chem. 193, 265-275. MCKENNA,
M. J., ZEMPEL,
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E. O., AND
GEHRING, P. J. (1978). The pharmacokinetics of (r4C) vinylidene chloride in rats following inhalation exposure. Toxicoi. Appl. Pharmacol. 45, 599-610. MILLER, G. L. (1959). Protein determination for large numbers of samples. Anal. Chem. 31, 964. OMURA, T., AND SATO, R. (1964). The carbon monoxide-binding pigment of liver microsomes. 1. Evidence for its hemoprotein nature. J. Biol. Chem. 239,237&2378. RECKNAGEL, P. O., and GLENDE, E. A. (1973). Carbon tetrachloride hepatotoxicity: An example of lethal cleavage. CRC Crit. Rev. Toxicol. 2, 263-279. SATO, A., NAKAJIMA, YAMA, N. (1975a).
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Kinetic studies on sex difference in susceptibility to chronic benzene intoxicationWith special reference to body fat content. Brit. J. Ind. Med. 32, 321-328. SATO, A., NAKAJIMA, T., AND FUJIWARA, Y. (1975b). Determination of benzene and toluene in blood by means of a syringe-equilibration method using a small amount of blood. Brit. J. Ind. Med. 32, 216214. SATO, A., AND NAKAJIMA, T. (1979). A vial-equilibration method to evaluate the drug-metabolizing enzyme activity for volatile hydrocarbons. Toxicol. Appl. Pharmacol. 41, 41-46. SLATER, T. F. (1966). Necrogenic action of carbon tetrachloride in the rats: A speculative mechanism based on activation. Nature (London) 209, 3&40. TANAKA, S., AND IKEDA, M. (1968). A method for determination of trichloroethanol and trichloroacetic acid in urine. Brit. J. Znd. Med. 25, 214219.
AND
APPLIED
PHARMACOLOGY
50,549-556
(1979)
Enhanced Activity of Liver Drug-Metabolizing Enzymes for Aromatic and Chlorinated Hydrocarbons following Food Deprivation T. NAKAJIMA AND A. SATO Department
of Hygiene, School of Medicine,
Shinshu University, 390 Japan
Asahi, Matsumoto,
Received January 16, 1979; accepted May 16, 1979
EnhancedActivity of Liver Drug-MetabolizingEnzymesfor Aromatic andChlorinated HydrocarbonsfollowingFood Deprivation.NAKAIIMA, T., AND SATO, A. (1979).Toxicol. Appl. Pharmacol. 50, 549-556. The in vitro activity of liver microsomal enzymesto metabblize somearomaticand chlorinatedhydrocarbonswasenhancedremarkablyin l-day fastedratsof bothsexes, althoughfastingproducednosignificantincrease in themicrosomal protein and cytochromeP-450 contents.Eventhe activity per wholeliver wasat a significantly increased level, despitethe fact that fastingcauseda markedlossof liver weight. A sexdifferencewasnotedin the metabolism of thesehydrocarbonsboth in fed and l-day fastedrats. However,food deprivationfor 3 dayswasfound to decrease the extentof this sex difference.A metabolismstudy usingtrichloroethyleneasa modelsubstrateshowed that the rate of metabolism in vivo wasalsoenhanced by fasting.
Food deprivation has been reported to produce a variety of substrate- and sex-related effects on liver drug-metabolizing enzymes in rats: After 72 hr it decreased aminopyrine and hexobarbital metabolism in male rats but increased them in female rats, whereas it increased the activity of aniline hydroxylase in both sexes (Kato and Gillette, 1965). It is also known that food deprivation potentiates the hepatotoxicity of some chlorinated hydrocarbons such as carbon tetrachloride (Krishnan and Stenger, 1966; Diaz G6mez et al., 1975; Jaeger et al., 1975), chloroform (Davis and Whipple, 1919; Goldschmidt et al., 1939), and l,l-dichloroethylene (Jaeger et al., 1973c, 1974, 1975; McKenna et al., 1978). However, very little has hitherto been known regarding the effects of food deprivation on the metabolism of these hydrocarbons.
We have developed recently a very simple method for evaluating the liver drug-metabolizing enzyme activity using a gas chromatographic technique (Sato and Nakajima, 1979), which can be applied to enzyme studies in vitro of almost all of the volatile hydrocarbons. In the present report, the effects of food deprivation on the in vitro and in vivo metabolism in rats of both sexesof some aromatic and chlorinated aliphatic hydrocarbons have been described. METHODS Animals. Wistar male and femalerats were fed
pellet food (Nippon Clea, Type E-2) and water ad Iibitum until they reacheda weight of 250g in
maleand 150g in femalerats.Onreachingthe weight, they were fed a nutritionally adequateliquid diet (1Cal/ml) as previously reported (DeCarli and Lieber, 1967).The diet was given daily at 4 PM,
549 All
0041-008X/79/12054948$02.00/0 Copyright Q 1979 by Academic Pres, Inc. rights of reproduction in any form reserved. Printed in Great Britain
550
NAKAJIMA
AND
80 ml to male and 70 ml to female rats, and rats were usually sacrificed at 10 AM for enzyme assay. The feeding was continued for at least 3 weeks before sacrifice. Food deprivation was caused by substituting an equivolume of tap water for the liquid diet. The period of time between feeding and sacrifice was an important factor affecting the activity of liver drug-metabolizing enzymes (unpublished observations). In this regard, the liquid diet feeding had an advantage in our metabolism study over the normal pellet food feeding; rats drank the liquid diet given at 4 PM almost completely by 10 PM on the same day. Chemicals. Nicotinamide-adenine dinucleotide phosphate disodium salt (NADP) and glucose-6phosphate disodium salt (G-6-P) were obtained from Boehringer-Mannheim, and the other chemicals used were of reagent grade purchased from Nakarai Chemicals, Kyoto. Metabolism study in vitro. Rats were decapitated followed by exsanguination. The liver was minced and a 10% (w/v) crude homogenate was made with 1.15 % KCI-O.01 M Na+/K+-phosphate buffer, pH 7.4, in a glass homogenizer. The homogenate was then centrifuged at 30,OOOg for 10min. A part of the resultant supernatant was further centrifuged at 105,OOOg for 60 min to obtain microsomal pellets. One milliliter of the 10,000g supernatant was normally used as the source of the enzyme. One-tenth of a milliliter of a water solution of hydrocarbons, mostly 5 to 10 ~1/100 ml, was used as the substrate. The enzyme and substrate were incubated with 2 ml cofactor solution (containing: 0.5 prnol NADP; 10 pmol G-6-P; 25 ,umol MgCI,) in an airtight vessel for 10 min. The rate of metabolic rection was assessed by measuring the rate of substrate disappearance and was generally expressed as nanomoles per gram of liver per minute (Sato and Nakajima, 1979).
Microsomal
protein
Fed l-Day fast 2-Day fast 3-Day fast
334.9 * 17.36 320.2k 10.0 298.8 k 8.0 297.5 f 9.5
Liver (9)
P-450
contents.
RESULTS EfSect of Food Deprivation on Body Weight, Liver Weight, and Microsomal Protein and Cytochrome P-450 Contents
After l-day fasting, male rats lost 5% and female rats 4% of their original body weights. Male rats had about 25% and female rats 20% less liver weight than the respective 1
ON BODY AND LIVER WEIGHTS
Male Body (g)
and cytochrome
The microsomal protein content was measured according to Lowry et al. (1951) as modified by Miller (1959). The amount of cytochrome P-450 was estimated by the method of Omura and Sato (1964). Effect of food deprivation on trichIoroethylene metabolism in vivo. In the in vivo metabolism study, trichloroethylene was selected as a model substrate, because the metabolites excreted in urine are not found normally in urine, thus urine analysis gives more accurate information about the effect of food deprivation. Four fed and four l-day fasted, male rats were exposed to 500 ppm of trichloroethylene for 4 hr (8 to 12 AM) in a dynamic airflow exposure chamber as previously reported (Sato et al., 1975a). After cessation of exposure the concentration of trichloroethylene in blood and the amount of its urinary metabolites (total trichloro compounds, TTC) were measured at varying times. Trichloroethylene concentration in blood was measured by a syringe-equilibration method (Sato et al., 1975b). Urinary TTC was estimated as described by Tanaka and Ikeda (1968). Statistical analysis. Results were compared by analysis of variance (Table 1, Fig. 1) or Student's t test (Tables 2 and 3).
TABLE EFFECTS OF FOOD DEPRIVATION
SAT0
Female Liver/body”
Body (8)
Liver 64 1.8
Liver/body”
9.74kO.73
2.9OkO.20
182.3+
6.2OkO.28
3.40&0.15
7.58kO.38
2.39kO.11'
178.7k8.8
5.08kO.23
2.84+0.02c
6.7OkO.26
2.24_+0.09c*d
162.5k4.8
4.25kO.37
2.61&0.07+'
6.17kO.06
2.07~0.08c~d~e
167.8k9.3
3.97kO.40
2.36~0.11c~d~e
0 Liver weight per 100 g body wt. b The values represent the mean f SD for five rats. c Significantly different from fed rats, p < 0.05. d Significantly different from l-day fasted rats, p x 0.05. e Significantly different from 2-day fasted rats, p < 0.05.
FASTING
ON HYDROCARBON
TABLE
551
METABOLISM
2
EFFECTSOF Foot DEPRIVATION ON LIVER MICROKIMAL PROTEIN AND CYT~CHROME P-450 C0m~Nm Male Animals
Protein (mg/g liver)
Fed l-Day fast 2-Day fast 3-Day fast
22.7 f 2.7” 23.0+ 2.7 22.0 + 2.0 22.3k2.4
Female
P-450 (nmol/mg protein)
Protein (mg/g liver)
0.842*0.123* 0.823 &-0.030b 0.782 f 0.042b 0.799 + 0.090*
22.5+ 23.7+ 23.6 + 22.1+
P-450 (nmol/mg protein)
1.5 1.7 2.4 0.7
0.638kO.051 0.673 + 0.044 0.633 i- 0.039 0.638 + 0.023
a The values represent the mean+ SD for five rats. b Significantly different from the respective female rats, p < 0.05. TABLE
3
EFFECT OF I-DAY Foot DEPRIVATION ON THE ACTIVITY OF DRUG-METABOLIZING ENZYMES” Metabolic rate (nmol/g/min) Male Substrates Benzeneb*c*d Tolueneb~c*d~e m-Xyleneb*c,d Ethylbenzeneb~c*d*e n-Propylbenzeneb*‘*@ Cumeneb*Csd@ Styreneb*C*d*e Dichloromethaneb*c*d*e Chloroform**‘** Carbon tetrachlorideb*c*dse 1, l-Dichloroethanec~d~’ 1,2-Dichloroethaneb*csd*e l,l,l-Trichloroethane’ 1,l ,2-Trichloroethaneb*c*d*e 1,1,1,2-Tetrachloroethanec*d 1,1,2,2-Tetrachloroethanebvcsd Trichloroethyleneb*‘sd Tetrachloroethylene’ 1, l-Dichloroethyleneb*c*d l-Chloropropaneb~c~d*e Monochlorobenzenec*d
Fed 13.7* 5.4 18.lk4.9 21.0* 5.5 22.9k6.3 46.5 + 12.8 36.6k4.3 28.5k4.3 28.5r1.5 19.7k2.6 1.9kO.2 19.lk3.3 23.6+ 1.1 0.5 + 0.2 21.Ok1.9 8.1 k2.0 13.3kO.6 18.9+ 7.4 0.5 f 0.3 31.1 k6.6 37.6+ 5.9 8.2? 1.6
Fasted 36.4+ 2.8 40.3 + 4.8 41.0* 1.9 40.8 + 3.5 77.2+ 17.4 75.9k 1.6 46.1+ 1.6 60.9+ 1.9 55.1 + 7.5 5.9kO.8 56.0& 2.4 59.8 + 3.1 1.2kO.2 56.0+ 3.0 32.8 + 8.4 40.0 f 2.4 57.0+ 1.9 1.950.3 67.3 k 6.6 lOO.Ok 5.2 35.5 f 6.3
Female Fasted/fed
Fed
2.7 2.2 2.0 1.8 1.7 2.1 1.6 2.1 2.8 3.1 2.9 2.5 2.4 2.7 4.0
8.5 + 0.2 10.6k4.1 16.3 f 3.6 13.5k4.5 24.7 It 5.9 14.6f 4.5 16.8 I? 3.2 21.5k5.5 15.3k6.8 1.1 kO.5 14.6+ 1.6 14.8rt4.1 0.4kO.3 12.9+ 1.3 6.9k2.7
29.3k1.2 29.9k1.6 33.3 + 3.5 27.8 + 1.8 50.8 -+ 3.9 26.9 + 3.5 36.1+ 6.5 51.7k2.9 39.3k2.5 4.520.3 48.5+ 12.2 53.3k2.9 0.8kO.5 46.8 k 8.3 26.9_+ 6.7
3.4 2.8 2.0 2.1 2.1 1.8 2.1 2.4 2.6 4.1 3.3 3.6 2.0 3.6 3.9
3.0 3.0 3.8 2.2 2.7 4.3
15.725.2 13.4k3.8 0.7kO.5 25.4k2.7 17.8k2.9 lO.O& 0.8
30.5k1.6 38.4k2.4 1.8+ 1.5 44.6+ 4.2 40.8 f 10.2 31.3k5.6
1.9 2.9 2.6 1.8 2.3 3.1
a The values in the columns Fed and Fasted represent the meanf SD for five rats. * Significantly different between male and female fasted rats, p -c0.05. c Significantly different between fed and fasted male rats, p <: 0.01. d Significantly different between fed and fasted female rats, p-c 0.01. e Significantly different between male and female fed rats, p -c 0.05.
Fasted
Fasted/fed
552
NAKAJIMA AND SAT0
control fed rats. When food deprivation was the metabolic rates of all the hydrocarbons continued over a period of 2 or 3 days, the studied were markedly elevated in animals of body and liver weights were further de- both sexes. With all the marked loss of liver creased. Although the loss during this period weight in fasted rats, the total liver enzyme was somewhat less conspicuous than the activity (activity/whole liver/unit time) in loss caused by l-day fasting, a significant de- these rats was still significantly higher than crease in liver weight per 100 g body wt was that in fed rats. noted with this prolongation of food deprivation. These findings indicate that the liver is Sex Deference in the Effect of Food Deprivation on Drug-Metabolizing Enzyme Activity more seriously affected by food deprivation than the total body weight would suggest As shown in Table 3, male fed rats meta(Table 1). bolized most of the hydrocarbons more On the other hand, food deprivation pro- rapidly than fed female rats. This sex differduced no obvious effect on the microsomal ence in enzyme activity became much more protein and cytochrome P-450 contents noticeable after 1 day of fasting. (Table 2). However, when food deprivation was conSex differences were found in body weight, tinued over a period of 2 or 3 days, no further liver weight, and cytochrome P-450 content, significant increase of the enzyme activity but not in microsomal protein content. occurred in male rats, whereas the activity in female rats tended to further increase Activation of Liver Drug-Metabolizing En- during this prolonged fast (Fig. 1). Consezymes by Food Deprivation quently, the sex difference in the enzyme Metabolic rates of hydrocarbons in vitro in activity, which was evidently observed both fed and fasted rats are listed in Table 3. in the well-fed and l-day fasted rats, became Clearly, the liver of rats of both sexes meta- less apparent with the prolongation of fasting. bolized 1,l , I-trichloroethane, tetrachloroAs the liver weight decreased with proethylene, and carbon tetrachloride much less longed food deprivation, the enzyme activity rapidly than the other hydrocarbons. per whole liver in rats of both sexes was When the liquid diet was replaced by an lowered during this term compared with the equivolume of water 1 day before sacrifice activity in l-day fasted rats. 70-
-*-MALE -.a.--FEMALE
8 00. ? $ 50e
TOLUENE
i
11
BENZENE
0-e
0
12
3
olza DURATION
OF
FAST.
DAYS
FIG. 1. Activity of liver drug-metabolizing enzymes in relation to duration of food deprivation. Each point represents the meanf SD for five rats. (*) Significantly different from l-day fasted female rats, p < 0.05.
FASTING
ON HYDROCARBON
553
METABOLISM
these reconstituted 10,000g supernatants. As is shown in Table 4, the enzyme activation following food deprivation is associated with changes in the microsomes, but not in the Since the 10,000g supematant was used as soluble fraction. the enzyme source, there arises a question of Efect of Food Deprivation on Apparent K, whether the enhanced activity by starvation and V,,,,, for Toluene and Trichloroethylene has been derived from changes in microsome Metabolism or soluble fraction. The 10,000g supematant was centrifuged at 105,OOOg for 1 hr and Figure 2 shows the double reciprocal plots divided into the soluble and microsomal of metabolic rates of toluene and trichlorofractions. The soluble fraction from a fed ethylene versus substrate concentrations. rat was added to the microsome from a Clearly, not only the maximum velocity (V,,J but also the Michaelis constant (K,) fasted rat, and conversely the soluble fraction from the fasted rat to the microsome from changed with food deprivation. The values the fed rat. The metabolic rates of toluene for K, and V,,, obtained from Fig. 2 were and trichloroethylene were measured with as follows, respectively: 3.3 PM and 16.7
Metabolic Rates of Toluene and Trichloroethylene Measured by Interchanging Microsome and Soluble Fraction between Fed and Fasted Rats
TABLE
4
METABOLIC RATES OF TOLUENE AND TRICHLOROETHYLENE INTERCHANGING MICR~SOME (M) AND SOLUBLE FRACTION FED AND FASTED RATS=
MEASURED BY (S) BETWEEN
Metabolic rate (nmol/g/min) Enzyme sources (M) (M) (M) (M)
from from from from
Toluene
fed rat+(S) from fed rat fed rat+(S) from fasted rat fasted rat + (S) from fasted rat fasted rat+(S) from fed rat
Trichloroethylene
17.4k2.2 17.2+ 1.8 42.2+ 3.3 4O.lk2.1
18.1k1.3 18.0+ 1.2 56.0+ 2.7 52.4k2.9
a The values represent the mean f SD of five measurements. -.-FED --*--
7 0.2-
FASTED
TOLUENE
s z i ,G 0.1.
0.2-
‘:
?‘ii
TRICHIDROETHYLENE
3 e /’
f 0,’
,
_/‘. /
p.,’
,’
,x3-.
l/s. ,uM-’
,<
O.l-
A’
/p
,A’
,.o. /D.--,’
‘/S.
AIM-’
FW 2. Double reciprocal plots of metabolic rate (u) versus substrate concentration represents the mean for three fed or three l-day fasted male rats. .
(s). Each point
554
NAKAJIMA
AND
nmol/g/min in fed rats and 12.5 ,UM and 41.7 nmol/g/min in fasted rats for toluene; 3.8 PM and 20.0 nmol/g/min in the fed rats and 16.7 ,UM and 55.6 nmol/g/min in the fasted rats for trichloroethylene. Effect of Food Deprivation on the Transfer of Trichloroethylene in Male Rats
The time course of trichloroethylene concentration in blood and that of cumulative urinary excretion of its metabolites (TTC) are shown in Figs. 3 and 4, respectively. The postexposure concentration of trichloroethylene in blood was significantly lower in fasted rats than in fed rats. On the other hand, the fasted rats excreted a much greater amount of TTC in 24 hr after start of exposure than the fed rats. Particularly, the amount of TTC excreted during the first 5 hr was much larger in fasted rats than in fed rats. DISCUSSION A l-day fast decreased the liver weight of rats by about 20% without significant changes in microsomal protein and cytochrome P-450 contents. However, the fasting caused a remarkable increase in the activity of liver microsomal drug-metabolizing enzymes; even the activity per whole liver was at a
SAT0
significantly increased level in spite of the marked loss of liver weight in the fasted rats (Tables 1 and 2). This activation of enzymes following fasting was certainly associated with some changes in the microsomal fraction but not related to a change in the concentration of microsomal protein or cytochrome P-450. These findings suggest that the enzyme activation by fasting may differ from the enzyme
0.01 1. 0
a 1
a 2
4
6
8
HOURS
FIG. 3. Time courses of trichloroethylene concentration in blood after 4-hr exposure to 500 ppm of trichloroethylene. Each point represents the mean + SD for four fed or four l-day fasted male rats.
POST - EXPOSURE
Ok+4 0
t 45
5
8
10
12
14
24
HOURS
FIG. 4. Cumulative amount of TTC excreted in urine during and after 4-hr exposure to 500 ppm of trichloroethylene. Each point represents the mean f SD for four fed or four l-day fasted male rats.
FASTING ON HYDROCARBON METABOLISM
induction of phenobarbital which causes an increase of microsomal protein and cytochrome P-450 content (Conney, 1967). However, the underlying mechanism of the enzyme activation is not yet clear. In the in uivo metabolism study with trichloroethylene, its concentration in blood following inhalation exposure was found to be much lower in fasted rats than in fed rats, hence suggesting that its concentration in liver would have been lower in the former. Nevertheless, the fasted rats excreted about 50% as much urinary TTC than did the fed rats. Therefore, it can be said with certainty that food deprivation increases the metabolism of trichloroethylene not only in vitro but also in uiuo. Food deprivation has been reported to affect the activity of liver drug-metabolizing enzymes in a sex-related manner: it decreases the metabolism of aminopyrine in male rats but increases in female rats, whereas it increases the metabolism of aniline in both sexes (Kato and Gillette, 1965). Although an apparent sex difference was found in the activity of enzymes to metabolize most of the hydrocarbons studied, food deprivation activated the enzymes almost to an equal extent in both sexes. Some chlorinated hydrocarbons have been known to cause more serious liver damage in fasted rats than in fed rats (Davis and Whipple, 1919; Krishnan and Stenger, 1966; Jaeger et al., 1973c, 1974, 1975; Diaz Gomez et al., 1975; McKenna et al., 1978). However, the biochemical mechanism for this enhanced susceptibility of fasted rats has not been fully elucidated. The hepatic necrosis due to carbon tetrachloride is caused not by the hydrocarbon itself but by its active metabolites produced in the liver (Butler, 1961; Slater, 1966; Recknagel and Glende, 1973). In our present investigation, l-day fasting of rats increased the in vitro metabolism of carbon tetrachloride by about three to four times, suggesting that fasting may lead to a greater production of the toxic intermediates and
555
hence to a more severe liver damage. This may offer a possible explanation for the increased carbon tetrachloride-induced hepatotoxicity observed in fasted rats. Brief or 1%hr fasting has also been known to increase the susceptibility of rats to the hepatotoxicity of chloroform (Davis and Whipple, 19 19) and 1,l -dichloroethylene (Jaeger et al., 1973c, 1974, 1975; McKenna et al., 1978). In this case, the decreased concentration of hepatic glutathione (GSH) is said to be responsible for the increased susceptibility in the fasted rats, since GSH is greatly involved in the detoxification pathway for these hydrocarbons (Jaeger et al., 1973a, 1974; Docks and Krishna, 1976; McKenna et al., 1978). It is also generally accepted that chloroform (Ilett et al., 1973) and l,l-dichloroethylene (Jaeger er al., 1974; Bartsch et al., 1975) are activated to respective proximate toxicants, although a possibility that 1,I-dichloroethylene itself causes hepatic injury cannot be denied (Carlson and Fuller, 1972; Jenkins ef al., 1972; Jaeger et al., 1973b). In our metabolism study in vitro, the metabolism of chloroform and l,l-dichloroethylene in the liver of food deprived rats increased about three-fold. Therefore, it can be proposed that the elevated metabolism following food deprivation may also play an important role in increasing the susceptibility of fasted rats to these hepatotoxic hydrocarbons. REFERENCES BARTSCH, H., MALAVEILLE, C., MONTESANO, R., AND TOMATIS, L. (1975). Tissue-mediated mutagenicity of vinylidene chloride and 2-chlorobutadiene in Salmonella typhimurium. Nutwe (London) 255, 641-643. BUTLER,
T. C. (1961). Reduction of carbon tetrachloride in uico and reduction of carbon tetrachloride and chloroform in vitro by tissues and tissue homogenates. J. Pharmacol. Exp. Ther. 134, 311-319. CARLSON, G. P., AND FULLER, G. C. (1972). Interaction of modifiers of hepatic microsomal drug metabolism and the inhalation toxicity of l,ldichloroethylene. Rex Commun. Chem. Pathol. Pharmacol.
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556
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A. H. (1967). Pharmacological implication of microsomal enzyme induction. Pharmacol. Rev.
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DAVIS, N., AND WHIPPLE, C. (1919). The influence of fasting and various diets on the liver injury effected by chloroform anesthesia. Arch. Znt. Med. 23, 612-633. DECARLI, L. M., AND LIEBER, C. S. (1967). Fatty liver in the rat after prolonged intake of ethanol with a nutritionally adequate liquid diet. J. Nutr. 91, 331-336. DfAz G~MEZ, M. I., DE CASTRO, C. R., DE FERREYRA, E. C., D’ACOSTA, N., DE FENOS, 0. M., AND CASTRO, J. A. (1975). Mechanistic studies on carbon tetrachloride hepatotoxicity in fasted and fed rats. Toxicol. Appl. Pharmacol. 32, 101-108. DOCKS, E. L., AND KRISHNA, G. (1976). The role of glutathione in chloroform-induced hepatotoxicity. Exp. Mol. Pathol. 24, 13-22. GOLDSCHMIDT,
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(1939). The influence of the foodstuffs upon the susceptibility of the liver to injury by chloroform, and the probable mechanism of their action. J. Clin. Invest. 18, 277-289. ILETT, K. F., REID, W. D., SIPES,I. G., AND KRISHNA, G. (1973). Chloroform toxicity in mice: Correlation of renal and hepatic necrosis with covalent binding of metabolites to tissue macromolecules. Exp. Mol. Pathol. 19, 215-229. JAEGER, R. J., CONOLLY, R. B., AND MURPHY, S. D. (1973a). Diurnal variation of hepatic glutathione concentration and its correlation with l,l-dichloroethylene inhalation toxicity in rats. Res. Commun. Chem. Pathol. Pharmacol. 6, 465471. JAEGER, R. J., TRABULUS, M. J., AND MURPHY, S. D. (1973b). Biochemical effects of l,l-dichloroethylene in rats: Dissociation of its hepatotoxicity from a lipoperoxidative mechanism. Toxicol. Appl. Pharmacol. 24, 457-467. JAEGER, R. J., CONOLLY, R. B., AND MURPHY, S. D. (1974). Effect of 18 hr fast and glutathione depletion on l,l-dichloroethylene-induced hepatotoxicity and lethality in rats. Exp. Mol. Pathol. 20, 187-198. JAEGER,
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(1975). Short-term inhalation toxicity of halogenated hydrocarbons. Effects on fasting rats. Arch. Environ. Health 30, 26-30. JAEGER, R. J., TRABULUS, M. J., AND MURPHY, S. D. (1973~). The interaction of adrenalectomy, partial adrenal replacement therapy, and starvation with hepatotoxicity and lethality after l,l-dichloro-
ethylene intoxication. Toxicol. Appl. Pharmacol. 25, 491. JENKINS, L. J., TRABULUS, M. J., AND MURPHY, S. D. (1972). Biochemical effects of l,l-dichloroethylene in rats: Comparison with carbon tetrachloride and 1,2-dichloroethylene. Toxicol. Appl. Pharmacol. 23, 501-510. KATO, R., AND GILLETTE, J. R. (1965). Effect of starvation on NADPH-dependent enzymes in liver microsomes of male and female rats. J. Pharmacol. Exp. Ther. 150, 279-284. KRISHNAN, N., AND STENGER, R. (1966). Effects of starvation on the hepatotoxicity of carbon tetrachloride. Amer. J. Pathoi. 49, 239-246. LOWRY, 0. H., ROSEBROUGH, N. J., FARR, A. L., AND RANDALL, R. J.,(1951). Protein measurement with the Folin phenol reagent. J. Biol. Chem. 193, 265-275. MCKENNA,
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GEHRING, P. J. (1978). The pharmacokinetics of (r4C) vinylidene chloride in rats following inhalation exposure. Toxicoi. Appl. Pharmacol. 45, 599-610. MILLER, G. L. (1959). Protein determination for large numbers of samples. Anal. Chem. 31, 964. OMURA, T., AND SATO, R. (1964). The carbon monoxide-binding pigment of liver microsomes. 1. Evidence for its hemoprotein nature. J. Biol. Chem. 239,237&2378. RECKNAGEL, P. O., and GLENDE, E. A. (1973). Carbon tetrachloride hepatotoxicity: An example of lethal cleavage. CRC Crit. Rev. Toxicol. 2, 263-279. SATO, A., NAKAJIMA, YAMA, N. (1975a).
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Kinetic studies on sex difference in susceptibility to chronic benzene intoxicationWith special reference to body fat content. Brit. J. Ind. Med. 32, 321-328. SATO, A., NAKAJIMA, T., AND FUJIWARA, Y. (1975b). Determination of benzene and toluene in blood by means of a syringe-equilibration method using a small amount of blood. Brit. J. Ind. Med. 32, 216214. SATO, A., AND NAKAJIMA, T. (1979). A vial-equilibration method to evaluate the drug-metabolizing enzyme activity for volatile hydrocarbons. Toxicol. Appl. Pharmacol. 41, 41-46. SLATER, T. F. (1966). Necrogenic action of carbon tetrachloride in the rats: A speculative mechanism based on activation. Nature (London) 209, 3&40. TANAKA, S., AND IKEDA, M. (1968). A method for determination of trichloroethanol and trichloroacetic acid in urine. Brit. J. Znd. Med. 25, 214219.