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Potentiation of CCl4 and CHCl3 hepatotoxicity and lethality by various alcohols
FUNDAMENTALANDAPPLIEDTOXICOLOGY15,429-440(1990)
Potentiation of CC& and CHCi3 Hepatotoxicity and Lethality by Various Alcohols’ SIDHARTHA D. RAY AND HARIHARA M. MEHENDALE~ Department
of Pharmacology and Toxicology, University of Mississippi Medical Center, Jackson, Mississippi 392164505
Received
October
16. 1989; accepted
April 26, 1990
Potentiation of CC& and CHC13 Hepatotoxicity and Lethality by Various Alcohols. RAY, D., AND MEHENDALE, H. M. (1990). Fundam. Appl. Toxicol. 15, 429-440. Various aliphatic alcohols potentiate the toxicity of a wide range of xenobiotics including several haloalkanes. The present series of experiments were designed to test: (i) whether a single subtoxic dose of alcohol can potentiate Ccl., and CHCls hepatotoxicity, and (ii) whether this potentiation leads to greater animal lethality. Selected members of a homologous series of straight chain alcohols were chosen for this study. Methanol, ethanol, isopropanol, t-butanol, pentanol, hexanol, octanol, decanol, and eicosanol at equimolar doses (10 mmol/kg) were tested in the present investigation. Each alcohol was administered orally to male Sprague-Dawley rats (175-250 g) 18 hr prior to a single oral administration of CC& or CHCI,. Liver injury was assessedby plasma transaminases (alanine aminotransferase, ALT; aspartate aminotransferase. AST) and histopathological examination of liver sections 24 hr after the halomethane treatment. None of these alcohols alone increased plasma ALT or AST significantly, whereas CC& or CHCI, administration to alcohol-treated animals resulted in significant elevation ofplasma transaminases. Eicosano1 (20-carbon alcohol) did not potentiate the toxicity of either halomethane. Methanol, ethanol, isopropanol, and decanol in combination with CCL, caused massive liver damage but failed to augment CC& lethality. t-Butanol, pentanol, hexanol, and octanol significantly decreased the LD50 of CC&. The hepatotoxic effects of CHCls were potentiated by all of the alcohols and the LD5Os were also decreased significantly. On a comparative basis, alcohol-potentiated CHCl, toxicity was greater than the toxicity of CC&. These findings indicate that even though halomethane liver injury might be potentiated by alcohols, the underlying mechanisms differ among alcohols since not all alcohols potentiate the lethal effects of these halomethanes. o 1990 Society S.
ofToxicology
Potentiation of haloalkane toxicity by various alcohols is well known (Kutob and Plaa, 1962; Cornish and Adefuin, 1967; Anders and Harris, 198 1; Ueng et al., 1983). There is a plethora of research papers dealing with alcohol + CC4 interaction studies, whereas the interaction of alcohol + CHC13 has only ’ Presented at the 28th Annual Conference ofthe Society of Toxicology, February 27-March 3, 1989, Atlanta, GA. (1989) Toxicologist 9,59. ’ To whom all correspondence and reprint requests should be addressed. 429
sparsely been investigated. Alcohols, CC&, CHC& , and many related solvents are extensively used in chemical manufacturing processes, for research purposes, and in many end products in the form of formulations and hence human exposure to these singly or in combination is possible. The main objective of the present study was threefold. We wished to investigate whether: (a) there is any potentiation of halomethane hepatotoxicity when a single subtoxic dose of a homologous series of alcohols is preadministered; (b) under such conditions, alcohol-potentiated liver injury is 0272-0590/90 $3.00 Copyright 0 I990 by the Society of Toxicology. All rights of reproduction in any form reserved.
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of any consequence to animal survival; (c) there is a structure-activity relationship with respect to the potentiation of toxicity of Ccl4 and CHC13 by the alcohols. In all the previous alcohol + CC4 or alcohol + CHC13 interaction studies, varying doses of alcohols were employed to examine the possibility of potentiated liver injury. In numerous studies, doses of alcohols employed were themselves toxic. For example, 50% of the LD50 of methanol, ethanol, or isopropanol was employed by Traiger and Plaa (197 1) and Cantilena et al. (1979) whereas Cornish and Adefuin (1967) used 40% of the LD50 of methanol, t-butanol, and amyl alcohol. Ueng et al. (1983) treated the rats chronically with ethanol. One drawback of such studies is that secondary and tertiary mechanisms may interfere with the primary mechanism(s) of interactions. Moreover, the possibility of toxic interaction at low concentrations of the interactants cannot be reliably predicted from such studies. Systematic studies with a homologous series of alcohols have not been reported. Moreover, while most investigators have measured one or more parameters of liver injury, the issue of whether the alcohol-potentiated liver injury leads to increased lethality has not been investigated. In most studies, the degree of potentiation was evaluated by: (i) estimating elevation in plasma transaminases as an indication of liver injury; (ii) histopathological examination of liver and/or (iii) covalent binding of “CC14-‘4CHC13 metabolites to liver macromolecules. In the present investigation, hepatotoxicity was estimated by the leakage of cytoplasmic enzymes, aspartate and alanine aminotransferases (AST, ALT),3 into plasma, and by histopathological examination of liver sections by light microscopy. Lethality was measured as LD50 of Ccl4 or CHC13 after oral administration of the respective halomethane alone or after ’ Abbreviations AST, aspartate Schiff reagent.
used: ALT, alanine aminotransferase; aminotransferase; PAS, periodic acid-
MEHENDALE
prior exposure to methanol, ethanol, isopropanol, t-butanol, pentanol, hexanol, octanol, decanol, and eicosanol at a single, nontoxic dose administered orally. MATERIALS
AND
METHODS
Animals and treatment. Male Sprague-Dawley rats weighing 175-250 g were purchased from Charles River breeding laboratories (Willmington, MA) and maintained on Purina Chow rodent diet and water ad libitum for 5-9 days prior to the experiments. They were divided into four groups: (i) alcohol and halomethane vehicle control (i.e., saline or corn oil): (ii) alcohol control, IO mmol/kg (in corn oil or saline) + corn oil; (iii) CC4 or CHCIs alone: saline or corn oil + Ccl4 or CHC13 (0. I, 0.5, or 1 ml/kg), one of the doses was administered as I : I solution in corn oil; and (iv) alcohol and halomethane combination: alcohol IO mmol/kg + Ccl, or CHCI,. Alcohols and halomethanes were administered po. Eighteen hours before the dose of a single administration of Ccl,, CHC&, or corn oil vehicle, the animals received a given alcohol or a suitable vehicle such as corn oil or saline. The alcohol pretreatment regimen (10 mmol/kg, po) was chosen based on preliminary experiments which showed that none of the alcohols employed in this study caused liver injury upon a single oral administration to rats. Alcohols at this dose did not cause any elevation in plasma enzymes nor did they elicit any overt histological changes in the liver. The animals were returned to their respective cages after the administration of the chemicals. All animals were housed in plastic cages over untreated corncob bedding in our central facilities at 25 f 1°C and 50-800/u relative humidity. The following depicts the treatment protocol employed in these studies: I Saline/corn
oil
I Alcohol
oil
Saline/corn
518 hr Saline/corn
118 hr
I Saline/corn
oil
118 hr
118 hr
CC14 or CHCls
CC& or CHCI,
L24 hr
./24 hr
Oil
L24 hr
./24 hr
Plasma enzymes
I Alcohol
& histopathology
of Liver.
Plasma enq’mes. Twenty-four hours after the administration of Ccl,. CHC&, or corn oil, the animals were lightly anesthetized with diethyl ether, and blood was collected with heparinized syringes. Plasma was separated by centrifugation ofblood, and the extent of liver damage was assessed by estimating the activities of AST and ALT in the plasma. The enzymes were measured according to the method of Reitman and Frankel (1957) using a Sigma kit (procedure No. 505). Histopathology. When the animals were killed under ether anesthesia, the livers were rapidly excised, rinsed in
ALCOHOL-POTENTIATED
c23000 3 ~2000 E 4 “E1000 z e 64
0 : Saline/ . : Methanol A : Ethanol
corn
CC& AND CHCll TOXICITY
oil
I
A: lsopropanol
0 : Saline/
corn
r : Hem’01 v: 0ctano1
431 b
oil
,m.,.r.nnl 6 : D,,....,. 0 : Eicosanol
0 : t-Butanol n : Pentanol ,/I *ye/; ; 1/- i+=yzs*
om-h
fi/
n
0
0
0.1
0.5
1.0
CC14
CC14
(ml/kg)
(ml/kg)
FIG. I. Effects of various alcohol pretreatments on CC&-induced elevation of plasma AST. Male S-D rats received a single oral administration of the indicated alcohol (10 mmol/kg) in either saline or corn oil vehicle. Eighteen hours later, they received a single oral administration of a solution of CCL, (0.1,0.5, or 1 ml/kg) in corn oil, or corn oil alone. Twenty-four hours later, the plasma transaminases were determined. The figure shows plasma AST activities for control and treated animals. For the sake of clarity, two panels (a and b) are used to show the results. Means f SEM of three to five individual determinations. An asterisk indicates that the value is significantly different from the controls (p c 0.05). With the exception of eicosanol. all alcohols potentiate the CC&-induced elevation of AST. Significance: Response from 0.1 ml CC&/ kg is significantly different from I ml CC&/kg, but not from 0.5 ml CC&/kg; 0.1 ml CC&/kg is significantly different from 0.1 ml CCWkg in combination with methanol, ethanol, isopropanol, t-butanol, pentanol. hexanol, or octanol. Similarly, 0.5 ml CC&/kg alone is significantly different from various alcohols + 0.5 ml CC&/kg combinations except decanol and eicosanol. Also, I ml CC&/kg alone is significantly different from 1 ml CC&/kg in combination with various alcohols, except eicosanol.
0.9% NaCl, blotted, and weighed. A portion of each liver was sliced and fixed in phosphate-buffered 4% formaldehyde and then embedded in paraffin. Sections were stained with either periodic acid-Schiff (PAS) reagent or hematoxylin and eosin for histopathological evaluation under a light microscope.
0: .:
Saline/ Methanol
corn
oil
0: w:
t-Butanol Pentand
”
” 0.5 CC14
Lefhalitv. For 1Cday LD50 determination, four groups of rats consisting of four rats per group were treated po with the indicated alcohol (10 mmol/kg). Eighteen hours later, either CC4 or CHCls was administered in a geometric progression such that one group received one of the four doses. The number of dead ani-
1.0
(ml/W
rc
0
0.1
0.5
1.0
CC14 (ml/kg)
FIG. 2. Effects of various alcohol pretreatments on CC&-induced elevation of plasma ALT. Treatment and other details are as indicated in Fig. 1. Results are means + SEM of three to five individual determinations. Unless otherwise stated, all the values are significantly different from the respective controls (p G 0.05). (b) Eicosanol increased liver injury only at the I ml CCL/kg dose level. Significance: Response from 0.1 ml CC&/kg is significantly different from 1 ml CC&/kg, but not from 0.5 ml CC&/kg alone; 0.1 ml CC&/kg is significantly different from 0.1 ml CC&/kg in combination with methanol or isopropanol, tbutanol, pentanol, hexanol, or octanol. Similarly, 0.5 ml CC&/kg alone is significantly different from various alcohols + 0.5 ml CC&/kg combinations except ethanol, decanol, and eicosanol. Also, I ml CCL, atone is significantly different from 1 ml CC&/kg in combination with various alcohols.
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RAY AND MEHENDALE
mals was recorded during two daily observations for 14 days, and the LD50 was determined according to the procedures of Thompson (1947) and Weil (I 952). Sixteen rats were employed for each of the alcohols used. For the LD50 determination of CCL, and CHCI, alone, the rats received corn oil vehicle 18 hr prior to the respective halomethane. Statistics. Statistical significance was determined by ANOVA followed by a Dunnett’s t test. Asterisks in the tables and figures indicate the statistical significance of the data points, and the levels of significance have been indicated in each table and figure legend. Significance of LD50 values was determined by 95% confidence limit intervals.
RESULTS 1. Changes in Plasma Enzymes Figure 1 shows the effects of various alcohol pretreatments on plasma AST levels. All alcohols employed except eicosanol increased plasma AST levels after the administration of Ccl.,. None of the alcohols alone increased AST levels significantly (data not shown). CC& alone, depending upon the dosage employed, increased AST levels significantly. However, the increase in AST levels in the alcohol + CC& group was severalfold higher than that with Ccl, alone, indicating a significant enhancement of liver injury. Among all the alcohols, t-butanol, methanol, and hexanol were more effective than the others in enhancing the Ccl,-induced serum transaminase elevations. Figure 2 shows the effects of various alcohol pretreatments on plasma ALT levels. The pattern of enhancement closely paralleled AST levels. With the exception of eicosanol, the rest of the alcohols increased ALT activity after the administration of CCL, indicating thereby that the combination treatment elicits severalfold greater toxicity when compared to either of the two agents individually. In this series, hexanol was found to be most potent, whereas methanol, isopropanol, t-butanol, and pentanol closely paralleled each other. Figure 3 shows the effect of various alcohol pretreatments on CHCl, liver injury as as-
sessed by plasma AST activity. All the alcohols increased serum AST levels after the administration of CHC& . However, the degree of increase is lower than that with the alcohol + CC& combination treatment. Eicosanol pretreatment did not result in any enhancement of CHCl,-induced plasma AST elevation. In contrast to the methanol + CCL, combination, the methanol + CHC13 combination was less effective in the elevation of the serum transaminases. Likewise, ethanol and isopropanol were weaker potentiators. Figure 4 shows the effect of various alcohol pretreatments on CHC& toxicity assessed by plasma ALT activity. It appears from the results that either alcohol or CHC13 when administered individually was incapable of increasing plasma ALT levels, whereas alcohol followed by a single dose of CHC& elicited significant liver injury as indicated by significantly elevated plasma ALT levels. However, eicosanol pretreatment failed to cause a CHC&-induced elevation of plasma ALT activity. Interestingly, decanol caused a greater potentiation of CHC13 toxicity in comparison to its effects on Ccl, toxicity. 2. Histopathology Liver sections were examined under a light microscope for the following signs of injury. Hepatocellular necrosis, swollen or ballooned cells, fatty infiltration and sinusoidal dilatation, inflammation, and infiltration by polymorphonuclear cells. Histopathological examination of the liver sections yielded findings which were consistent with the plasma transaminase data. Figure 5a shows the photomicrographs of PAS-stained liver sections from variously treated rats. Control (top left; saline + corn oil treatment): the photomicrograph shows normal architecture ofthe liver. CCL, (0.5 ml/ kg) elicited very limited centrilobular necrosis (top right) accompanied by findings typical of CCL, hepatotoxicity. Hexanol (hexanol + corn oil; bottom left) did not cause any sig-
ALCOHOL-POTENTIATED 0 : Saline/ 0 : Yethmol A : Ethanol
Corn
433
Ccl, AND CHC& TOXICITY
a
oil
; ?I I
l* A* A* 0 0.5
0.1
CHC13
1.0
0
0.1
(ml/kg)
0.5
CHC13
1.0
(ml/kg)
FIG. 3. Effects of various alcohol pretreatments on CHCl,-induced elevation of plasma AST. Male S-D rats received a single oral administration of the indicated alcohol (10 mmol/kg) in either saline or corn oil vehicle. Eighteen hours later, they received a single oral administration of 1:1 solution of CHC& (0.1,0.5, or 1 ml/kg) in corn oil or corn oil alone. Twenty-four hours later, the plasma transaminases were determined. The figure shows plasma AST activities for controls and treated animals. For clarity, two panels (a and b) are used to show the results. Results are means + SEM of three to five individual determinations. Unless otherwise stated, all the values are significantly different from the control (p G 0.05). With the exception of eicosanol, all alcohols potentiated the CHClr-induced elevation of AST. Significance: Response from 0.1 ml CHC&/kg is significantly different from 1 ml CHC&/kg, but not from 0.5 ml CHCIJ kg; 0.1 ml CHC13/kg alone is significantly different from 0.1 ml CHC& in combination with methanol, tbutanol, pentanol, hexanol, octanol, or decanol. Similarly, 0.5 ml CHClJkg alone is significantly different from various alcohols + 0.5 ml CHCls/kg combinations except ethanol and eicosanol. Also, 1 ml CHCls/kg alone is significantly different from all the alcohols in combination with 1 ml CHClJkg, except eicosanol.
nificant liver injury except marginal fatty infiltration and sinusoidal dilatation. The hexano1 + CC& (0.5 ml/kg) combination treatment (bottom right) caused massive hepatic
r;;1500 5 g
r a^^,.
0 : Saline/ corn 0: YethanoI A : Ethanol
injury with the following characteristics: (i) centrilobular necrosis, typical of massive CCL+ injury; (ii) ballooned hepatocytes in the centrilobular and midzonal areas; (iii) mas-
oil
A: Isopropanol t-Butan : Pentan
0 CHC13
(ml/kg)
0.1
0.5 CHC13 (ml/kg)
1.0
---
FIG. 4. Effects of various alcohol pretreatments on CHCl,-induced elevation of plasma ALT. Treatments and other details are as indicated in Fig. 3. Results are means ? SEM of three to five individual determinations. Unless otherwise stated, all of the values are significantly different from the respective controls (p G 0.05). Eicosanol did not potentiate liver injury. Significance: Response from 0.1 ml CHCls/kg is significantly different from 1 ml CHC&, but not from 0.5 ml CHCls/kg; 0.1 ml CHCl&g alone is significantly different from 0.1 ml CHClJkg in combination with methanol, isopropanol, t-butanol, hexanol, octanol, or decanol. Similarly, 0.5 ml CHCl&g alone is significantly different from all the alcohols in combination with 0.5 ml CHCls/kg, except ethanol and eicosanol. Also, 1 ml CHClJkg alone is significantly different from all the alcohols in combination with 1 ml CHClr/kg, except eicosanol.
434
RAY
AND
MEHENDALE
ALCOHOL-POTENTIATED
sive depletion of glycogen, (iv) increased infiltration of inflammatory cells; and (v) dead cell debri scattered over a significant portion of the hepatic lobule. All of these findings were typical of livers of all animals receiving the hexanol + CCL, combination treatment. The same qualitative morphological alterations were evident in hexanol-pretreated rats receiving CC& at a dose of either 0.1 or 1 ml/kg. The degree of the injury seen in these livers paralleled the Ccl, dose administered. The histopathological findings described above typify all of the alcohol + Ccl, combination treatments. Neither alcohol treatments alone nor CC& at 0.1 ml/kg alone elicited any significant effects. The most significant effects of treatment with alcohol alone were sinusoidal dilatation and lipid accumulation. The effects of hexanol (Fig. 5a, bottom left) represent the worst case in this regard. CCL, alone at 0.5 ml/kg caused greater injury typified by the histological findings associated with extensive hepatolobular necrosis, ballooned cells, fatty infiltration, and infiltration of polymorphonuclear cells. Hexanol potentiated CC& toxicity to the greatest extent in comparison to all of the other alcohols. Eicosanol did not increase the toxicity of CCL, at any of the three doses of the latter employed in this study. The other alcohols potentiated liver injury to varying degrees in the general order of hexanol > tbutanol > pentanol > octanol % isopropanol > decanol > methanol > ethanol, with respect to histopathological alterations in the liver (data not shown). Figure 5b shows the liver histopathological findings of the toxic interaction between octanol and chloroform. Treatment with either chloroform (0.5 ml/kg) or octanol(l0 mmol/ kg) alone did not show any significant liver
CC& AND CHQ
435
TOXICITY TABLE 1
EFFECT OFVARIOUSALCOHOISON CC& LETHALITY Alcohol
LD50
95% Confidence limits
Control Methanol Ethanol Isopropanol t-Butanol Pentanol Hexanol Octanol Decanol
4.50 2.78 3.36 2.99 1.06* 1.OP 0.75s 1.49* 3.00
3.10-6.48 2.02-3.84 2.38-4.75 2.01-4.47 0.65-I .06 1.06-1.06 0.46- 1.22 1.00-2.23 2.01-4.47
Note. The alcohols were administered po as a 25% solution in either corn oil or saline, I8 hr prior to the administration of CC&. The LD50 values were determined by the method of Thompson (1947) and Weil(1952) after a 14-day observation. An asterisk indicates that the value is significantly lower than the LD50 of Ccl, alone (p G 0.05).
injury except for some fatty infiltration in livers of rats receiving CHC13 alone. However, the octanol + CHC13 combination treatment caused massive damage identical to the hexano1 + CCL, treatment. Hepatic injury was evident as follows: (i) ballooned hepatocytes in the centrilobular and midzonal areas; (ii) massive depletion of glycogen; (iii) extensive necrotic cells in the centrilobular and midzonal areas; (iv) fatty infiltration throughout the lobule: and (v) infiltration of polymorphonuclear cells in areas of hepatocellular necrosis. The interaction of octanol + CHQ depicted in Fig. 5b represents the most severe interaction among all alcohols employed in this study. Eicosanol did not enhance the toxicity of CHC& at any dose of the halomethane. Other alcohols caused enhancement of liver injury
FIG. 5. (a) All fields show PAS-stained liver sections from male S-D rats (10 m. X90): (top left) control; (top right) CC4 (0.5 ml/kg); (bottom left) hexanol(l0 mmol/kg); and (bottom right) hexanol(l0 mmol/ kg) + CC& (0.5 ml/kg). Massive liver damage is evident in the liver section from hexanol + CC&-treated rats. (b) All fields show PAS-stained liver sections from male S-D rats (10 m, X90): (top left) control; (top right) CHCI, (0.5 ml/kg): (bottom left) octanol (10 mmol/kg); and (bottom right) octanol (IO mmol/kg) + CHC& (0.5 ml/kg). Massive damage is evident in the liver section from octanol + CHCl,-treated rats.
436
RAY AND MEHENDALE
to a varying degree. In general, the order ranking for the enhancement of CHC13 liver injury was as follows: hexanol > octanol > isopropanol L t-butanol > decanol > methanol > ethanol (data not shown).
3. Lethality Eflects of various alcohol pretreatments on the LDSO of CC&. These studies were conducted in order to determine if alcohol-potentiated CC& liver injury was of any consequence to animal survival. The findings (Table 1) reveal rather interesting observations in this regard. Although a number of alcohols are capable of potentiating hepatotoxicity, not all alcohols which potentiate hepatotoxic effects increased the lethal effects. The alcohols fell into three groups: the first group of alcohols, which potentiates liver injury of Ccl, but not CC4 lethality significantly, is represented by methanol, ethanol, isopropanol, and decanol. The second group of alcohols, which potentiates liver injury and also the lethal effects of CC& as evidenced by the decreased LD50 of CCL, comprises t-butanol, pentanol, hexanol, and decanol, t-butano1 being the most potent in this regard. The third group is represented by eicosanol, which potentiated neither the hepatotoxic nor the lethal effects of CC& (Table 1). Eflects of various alcohol pretreatments on the LD50 of CHClj. Table 2 shows the LD50 values of CHC13 and the alcohol + CHC13 treatment. The alcohol + CHC13 interaction resulted in greater lethality than the alcohol + CC& interaction. All of the alcohol pretreatments significantly decreased the LD50 of CHC13 in contrast to the alcohol + CC4 interaction. Octanol caused the maximal effects followed by t-butanol and isopropanol. Methanol caused the least decrease in the LD50 of CHCl3. On a comparative basis, the alcohol + CCL combination treatment did cause severe hepatotoxicity as indicated by the severalfold higher plasma transaminase levels. It appears
TABLE 2 EFFECT OF VARIOUS ALCOHOLS ON CHClr LETHALITY Alcohol
LD50
95% Confidence limits
Control Methanol Ethanol Isopropanol t-Butanol Pentanol Hexanol Octanol Decanol
1.99 0.84* 0.84* 0.501 0.50* 0.70* 0.63* 0.40* 0.80*
1.34-2.98 0.50-1.22 0.60-1.19 0.31-0.82 0.26-0.96 0.43-1.15 0.30-1.29 0.27-0.59 0.54-1.19
Note. The alcohols were administered po as a 25% solution in either corn oil or saline, 18 hr prior to the administration of CHCl,. The LD50 values were determined by the method of Thompson (1947) and Weil ( 1952) after a 14-day observation. An asterisk indicates that the value is significantly lower than the LD50 of CHC& alone (p c 0.05).
that the degree of potentiation alcohol + CHC13 combination and leads to greater lethality.
elicited by the is more potent
DISCUSSION Results from the present series of experiments provide some new and interesting information on alcohol-potentiated halomethane toxicity. In general, the alcohols fell into three categories in the case of alcohol + Ccl, interaction. However, the alcohols could be categorized into two groups in the case of alcohol + CHC13 interaction. These findings serve to illustrate that generalization from alcohol interaction studies with one halomethane may not be applicable to another halomethane. With the alcohol + Ccl4 interaction, group (i) consisting of methanol, ethanol, isopropanol, and decanol potentiated liver injury but did not affect animal survival significantly. Group (ii) consisting of t-butanol, pentanol, hexanol, and octanol potentiated liver injury and also significantly decreased animal sur-
ALCOHOL-POTENTIATED
vival, whereas group (iii) represented by eicosanol potentiated neither liver injury nor lethality. Results from the alcohol + CHC& interaction studies are somewhat different. Alcohols fell into two distinct categories: group (i) methanol, ethanol, isopropanol, t-butanol, pentanol, hexanol, octanol, and decanol potentiated liver injury and significantly affected animal survival, whereas group (ii) represented by eicosanol, failed to interfere with either of the two events, hepatotoxicity or lethality. The structure-activity relationship of alcohols to their potential for the toxic interaction with halomethanes is of some interest in predictive toxicology. It may be suggested from the present series of experiments that (i) the degree of potentiation is dependent on the number of carbon atoms on the straight chain alcohols and (ii) hepatotoxicity eventually leads to lethality during this toxic interaction at least with alcohols from 4 to 8 carbons in the case of CC& and from 1 to 10 carbons in the case of CHC&. No clear understanding of the mechanism of alcohol + CC& or alcohol + CHC& potentiation has developed (Lowery et al., 1981). The maximal potentiating effect is not seen in vivo until 18 hr after the administration of the alcohols (Traiger and Plaa, 197 I), when the alcohols would have dropped to undetectable levels in the blood. It was postulated that alcohols trigger a time-dependent series of events responsible for the potentiation of halomethane toxicity. The observation that isopropanol was ineffective in potentiating 14CC14binding when added directly to microsomes metabolizing CC4 (Sipes et al., 1973) is consistent with this concept. Various mechanisms have been proposed such as enhanced absorption in the intestine (Lamson et al., 1928) increased hepatomicrosomal cytochrome P450-mediated metabolism (Scholler et al., 1970; Ilett et al., 1973; Anders and Harris, 198 I), depletion of liver glutathione (Estler and Ammon, 1966; MacDonald, 1977), increased lipid peroxidation (Maling
CC&
AND
CHC&
TOXICITY
437
et al., 1975), and a shift to hepatic hypermetabolism (Strubelt, 1978). The cascade of events during this toxic interaction and their precise role in the potentiation of liver injury remain elusive. The present series of experiments corroborate with several earlier reports: (i) Anders and Harris (1981) reported that 6.4 mmol isopropanol pretreatment did not affect the LD50 of Ccl4 significantly although metabolism was increased, and (ii) Reynolds et al. ( 1982) have reported a severalfold increase in CHC& metabolism after isopropanol pretreatment when compared to CC14. Our present findings that isopropanol potentiates hep atotoxic and lethal effects of CHC& and only potentiates hepatotoxic effects of CC& are consistent with these earlier reports. The marked ability of isopropanol to potentiate CC& hepatotoxicity has been demonstrated by various workers (Comish and Adefuin, 1967; Harris and Anders, 198 1; Ueng et al., 1983). Isopropanol appeared to enhance selectively the activity of one or more forms of cytochrome P450 which catalyze the oxidation of aniline and 7-ethoxycoumarin. The metabolism of ethyl morphine and benzo[a]pyrene was not affected by pretreatment with isopropanol. These results lend credence to the hypothesis that the cytochrome(s) affected by isopropanol may cause significant biotransformation of Ccl4 to an active metabolite, possibly phosgene, which causes a nonspecific destruction of cytochrome P450dependent monooxygenases (Ueng et al., 1983). While such mechanisms may offer an explanation for enhanced liver injury of CCL, upon isopropanol pretreatment, the reason for the lack of potentiated lethality is not apparent from such mechanisms. CHCl, is one of the metabolic intermediates of cytochrome P450-mediated CC& metabolism and is also known to be more toxic than Ccl,. It is well established that the hepatotoxic effects of CHC& are mediated by phosgene, a reactive intermediate metabolite of CHC13. Therefore, it would be reasonable to speculate that the mechanism by which a
438
RAY
AND
MEHENDALE
compound potentiates CHC13 hepatotoxicity might be dependent on either the increased phosgene production or diminished phosgene detoxification. Cianflone et al. (1980) have published the view that, above all, organelle susceptibility or other factors may play a significant role in the mechanism of potentiation. Purushotham et al. (1988) have identified one such factor in their recent studies. Mehendale et al. (1989) have proposed that an important determinant of the extent of liver injury is whether hepatocellular regeneration and hepatolobular restoration takes place. Thus, they are of the opinion that normally when limited liver injury occurs due to the widely accepted putative mechanisms upon the administration of a low dose of CHC13, the liver responds by stimulating hepatocellular regeneration and hepatolobular tissue repair (Mehendale et al., 1989; Mehendale, 1989a). Unless some event interferes with these restorative biological events, normally the animals are able to overcome such injury. A similar mechanism has also been advanced for CC& (Mehendale, 1989ab). In the present study, we have observed all alcohols with the exception of eicosanol to potentiate liver injury of CC& and CHC13. However, only a few of the alcohols potentiated lethal effects of Ccl, and most of them potentiated CHC13 lethality. Although speculative, it is tempting to rationalize these findings based on the following sequence. Pretreatment with alcohol increases the bioactivation systems for these halomethanes and initiation of liver injury occurs via the usual, widely accepted putative mechanisms. For those alcohols which did not increase the lethal effect of CC&, the liver tissue was able to respond by stimulating the tissue repair mechanisms, consisting of the stimulation of hepatocellular regeneration and hence hepatolobular restoration. At later time points, after most of the administered CC& has been exhaled, no additional injury occurs. With the tissue repair processes being stimulated, the limited injury is overcome in due time, as the newly divided cells replace the dead cells,
resulting in restoration of hepatolobular architecture, culminating in the complete recovery of the animals. Although these concepts will require experimental scrutiny and verification, our results with methanol, ethanol, isopropanol, and decanol in combination with CCL, are consistent with this sequence of events. A noteworthy point here is that higher doses of alcohols are required to potentiate CC& or CHC13 toxicity in comparison to the remarkable potentiation by chlordecone. Increased renal injury as a potential contributing factor for alcohol potentiation of CHC13 lethality (Hewitt et al., 1979) is a possible factor for consideration. We employed rats for our present studies. Renal toxicity of CHC13 has been reported in mice (Hewitt et al., 1979; Pohl, 1979; Zimmerman and Norbach. 1980; Smith and Hook, 1984). Potentiation of renal toxicity by CHC13 is not known to readily occur in the rat model (Kluwe, 198 1). Therefore, any significant contribution of renal toxicity in the present CHC13 lethality studies is unlikely. For the alcohols, which increased the lethality of Ccl, in addition to the liver injury, the same putative mechanisms are involved in initiating liver injury. However, in this case, the biological events leading to stimulation of hepatocellular division and tissue repair are impaired by mechanisms hitherto not well understood. The result is a progression of what is normally a recoverable liver injury leading to hepatic failure to a level inconsistent with survival. The extent of interference with the tissue repair processes is a function of dose response both with respect to the alcohol pretreatment and the halomethane treatment and also with respect to the specific halomethane. Thus, with CHC13 interaction, most of the alcohols cause a greater potentiation of liver injury and hence a greater potentiation of lethality. This is consistent with the known greater toxicity of CHC13 at comparable doses of CC4 and CHC13. These conceptual proposals are based on experimental evidence indicating that up to a limit, the degree of liver injury results in
ALCOHOL-POTENTIATED
the stimulation of a proportionate degree of tissue repair processes (Mehendale et al., 1989). Second, it has also been demonstrated that chemicals such as chlordecone, mirex, and phenobarbital differ in their potential for interfering with the stimulation of hepatocellular regeneration and tissue repair during interaction with Ccl, and CHC13 (Purushotham et al., 1988; Mehendale, 1989a,b, 1990; Mehendale et al., 1989).
CC&
AND
These studies were supported by a grant from the Air Force Office of Scientific Research (AFOSR-88-0009). Harihara M. Mehendale is the recipient of the 1988 Burroughs Wellcome Toxicology Scholar Award.
REFERENCES
ANDERS, M. W., AND HARRIS, R. N. (1981). Effect of 2-propanol treatment on carbon tetrachloride metabolism and toxicity. Adv. Exp. Biol. Med. 136, 59 l602.
CANTILENA, L. R., JR., CAGEN, S. Z., AND KLAASSEN, C. D. (1979). Methanol potentiation of carbon tetrachloride-induced hepatotoxicity. Proc. Sot. Exp. Biol. Med.
162,90-95.
CIANFLONE, D. J., HEWITT, W. R., VILLENEUVE, D. C.. AND PLAA, G. L. (1980). Role of biotransformation in the alterations of chloroform hepatotoxicity produced by kepone and mirex. Toxicol. Appl. Pharmacol. 53, 140-149. CORNISH, H. H., AND ADEFUIN, J. (1967). Potentiation of carbon tetrachloride toxicity by aliphatic alcohols. Arch.
Environ.
Health
14,447-449.
ESTLER, C. J., AND AMMON, H. P. T. (1966). Enzyme in der leber Weisser Mause nach einmaliger alkoholgabe. Med.
Pharm.
Exp.
l&299-305.
HEWITT, W. R.. MIYAJIMA. H., COTE, M. G., AND PLAA, G. L. (1979). Acute alteration of chloroform-induced hepato and nephrotoxicity by mirex and Kepone. TOXicol. Appl. Pharmacol.
43,509-527.
ILEUM, 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. 19,2 15-220. KLUWE, W. M. (198 1). The nephrotoxicity oflow molecular weight halogenated alkane solvents, pesticides. and chemical intermediates. In Toxicology ofthe Kid-
TOXICITY
439
nqv (J. B. Hook, Ed.), pp. 180-226. Raven Press, New York. KUTOB, S. D., AND PLAA. G. L. (1962). The effect of acute ethanol intoxication on chloroform-induced liver damage. J. Pharmacol. Exp. Ther. 96,99- 113. LAMSON, P. D.. MINOT, A. S., AND ROBBINS, B. H. (I 928). The prevention and treatment of Ccl, intoxication. J. Amer. Med. Assoc. 90,345-349. LOWERY, K., GLENDE, E. A., JR., AND RECKNAGLE, R. 0. (198 1). Failure of ethanol or isopropanol pretreatment to affect carbon tetrachloride-induced inhibition of hepatic microsomal calcium pump activity. Drug
ACKNOWLEDGMENTS
CHC13
Chem.
Toxicol.
4,263-273.
MALING, H. M., STRIPP. B., SIPES,I. G., HIGHMAN, B., SAUL, W., AND WILLIAMS, M. A. (1975). Enchanced hepatotoxicity of Ccl,, thioacetamide, and dimethylnitrosamine by pretreatment of rats with ethanol and some comparisons with potentiation by isopropanol. Toxicol. Appl. Pharmacol. 33,29 l-308. MACDONALD, C. M.. Dow, J., AND MOORE. M. R. (1977). A possible protective role for sullhydryl compounds in acute alcoholic liver injury. Biochem. Pharmacol. 26, 1529-1533. MEHENDALE. H. M. (1989a). Amplification of hepatotoxicity and lethality of CCL, and CHCl, by chlordecone. Rev. Biochem. Toxicol. 10,9 1- 138. MEHENDALE, H. M. (1989b). Mechanism oflethal interaction of chlordecone and Ccl4 at nontoxic doses. Toxicol. Lett. 49,2 1 l-249. MEHENDALE, H. M. (1990). Potentiation of halomethane hepatotoxicity by chlordecone: A hypothesis for the mechanism. Med. Hypotheses 32, in press. MEHENDALE, H. M., PURUSHOTHAM, K. R., AND LOCKARD, V. G. (1989). The time-course of liver injury and ‘H-thymidine incorporation in chlordeconepotentiated CHClz hepatotoxicity. Exp. Mol. Pathol. 51,3 l-47.
POHL, L. R. (I 979). Biochemical toxicology of chloroform. Rev. Biochem. To.rirol. 1,79-107. PURUSHOTHAM, K. R., LCKXARD, V. G., AND MEHENDALE, H. M. (1988). Amplification of chloroform hepatotoxicity and lethality by dietary chlordecone in mice. Toxicol. Pathol. 16,27-34. REITMAN, S., AND FRANKEL, S. (1957). A calorimetric method for the determination of serum oxaloacetic and glutamic pyruvic transaminase. Amer. J. Pathol. 28,56-63.
REYNOLDS, E. S., MOSLEN, M. T., AND TREINEN, R. J. ( 1982). Isopropanol enhancement of carbon tetrachloride metabolism in vivo. Life Sci. 31,66 l-669. SCHOLLER, K. L.. MULLER, E., AND PHEHWE, U. V. ( 1970). Verstarkung and unterdruckung der toxizitat von chloroform fur die leber durch pharmaka. Arzeneim. Forsch.
t&289-292.
SIPES, I. G.. STRIPP, B., KRISHNA, G., MALING, H. M., ANDGILLETTE. J. R. (1973). Enhanced hepatic micro-
440
RAY
AND
somal activity by pretreatment of rats with acetone or isopropanol. Proc. Sot. Exp. Biol. Med. 142,237-240. SMITH. J. H.. AND HOOK, J. B. (1984). Mechanism of chloroform nephrotoxicity III. Renal and hepatic microsomal metabolism of chloroform in mice. Toxicol. Appl. Pharmacol.
13,5 1 l-524.
STRUBELT, 0.. OBERMEIER. F.. SIEGERS, C. P., AND VOLPER, M. (1978). Increased CCll hepatotoxicity after low level ethanol consumption. To.xicology 10, 261-210.
THOMPSON, W. R. (1947). Use of moving average and interpolation to estimate median effective dose. Bacferiol. Rev. 11, 115-145. TRAIGER, G. T., AND PLAA, G. L. ( 197 1). Differences in the potentiation of carbon tetrachloride in rats by
MEHENDALE
ethanol and isopropanol pretreatment. Toxicol. Pharmacol. UENG,
Appl.
71,204-214.
T. H., MOORE,
L., ELVES,
R. G., AND
ALVARES.
A. P. (1983). Isopropanol enhancement ofcytochrome P-450-dependent monooxygenase activities and its effects on carbon tetrachloride intoxication. Toxical. AppI. Pharmacol.
71,204-214.
WEIL, C. S. (1952). Tables for convenient calculation of median effective dose (LD50 and ED50) and instructions in their use. Biometrics 8,249-263. ZIMMERMAN, S. W., AND NORBACH, D. H. (1980). Nephrotoxic effects of long-term carbon tetrachloride administration in rats. Arch. Pathol. Lab. Med. 104, 94-99.
Potentiation of CC& and CHCi3 Hepatotoxicity and Lethality by Various Alcohols’ SIDHARTHA D. RAY AND HARIHARA M. MEHENDALE~ Department
of Pharmacology and Toxicology, University of Mississippi Medical Center, Jackson, Mississippi 392164505
Received
October
16. 1989; accepted
April 26, 1990
Potentiation of CC& and CHC13 Hepatotoxicity and Lethality by Various Alcohols. RAY, D., AND MEHENDALE, H. M. (1990). Fundam. Appl. Toxicol. 15, 429-440. Various aliphatic alcohols potentiate the toxicity of a wide range of xenobiotics including several haloalkanes. The present series of experiments were designed to test: (i) whether a single subtoxic dose of alcohol can potentiate Ccl., and CHCls hepatotoxicity, and (ii) whether this potentiation leads to greater animal lethality. Selected members of a homologous series of straight chain alcohols were chosen for this study. Methanol, ethanol, isopropanol, t-butanol, pentanol, hexanol, octanol, decanol, and eicosanol at equimolar doses (10 mmol/kg) were tested in the present investigation. Each alcohol was administered orally to male Sprague-Dawley rats (175-250 g) 18 hr prior to a single oral administration of CC& or CHCI,. Liver injury was assessedby plasma transaminases (alanine aminotransferase, ALT; aspartate aminotransferase. AST) and histopathological examination of liver sections 24 hr after the halomethane treatment. None of these alcohols alone increased plasma ALT or AST significantly, whereas CC& or CHCI, administration to alcohol-treated animals resulted in significant elevation ofplasma transaminases. Eicosano1 (20-carbon alcohol) did not potentiate the toxicity of either halomethane. Methanol, ethanol, isopropanol, and decanol in combination with CCL, caused massive liver damage but failed to augment CC& lethality. t-Butanol, pentanol, hexanol, and octanol significantly decreased the LD50 of CC&. The hepatotoxic effects of CHCls were potentiated by all of the alcohols and the LD5Os were also decreased significantly. On a comparative basis, alcohol-potentiated CHCl, toxicity was greater than the toxicity of CC&. These findings indicate that even though halomethane liver injury might be potentiated by alcohols, the underlying mechanisms differ among alcohols since not all alcohols potentiate the lethal effects of these halomethanes. o 1990 Society S.
ofToxicology
Potentiation of haloalkane toxicity by various alcohols is well known (Kutob and Plaa, 1962; Cornish and Adefuin, 1967; Anders and Harris, 198 1; Ueng et al., 1983). There is a plethora of research papers dealing with alcohol + CC4 interaction studies, whereas the interaction of alcohol + CHC13 has only ’ Presented at the 28th Annual Conference ofthe Society of Toxicology, February 27-March 3, 1989, Atlanta, GA. (1989) Toxicologist 9,59. ’ To whom all correspondence and reprint requests should be addressed. 429
sparsely been investigated. Alcohols, CC&, CHC& , and many related solvents are extensively used in chemical manufacturing processes, for research purposes, and in many end products in the form of formulations and hence human exposure to these singly or in combination is possible. The main objective of the present study was threefold. We wished to investigate whether: (a) there is any potentiation of halomethane hepatotoxicity when a single subtoxic dose of a homologous series of alcohols is preadministered; (b) under such conditions, alcohol-potentiated liver injury is 0272-0590/90 $3.00 Copyright 0 I990 by the Society of Toxicology. All rights of reproduction in any form reserved.
430
RAY
AND
of any consequence to animal survival; (c) there is a structure-activity relationship with respect to the potentiation of toxicity of Ccl4 and CHC13 by the alcohols. In all the previous alcohol + CC4 or alcohol + CHC13 interaction studies, varying doses of alcohols were employed to examine the possibility of potentiated liver injury. In numerous studies, doses of alcohols employed were themselves toxic. For example, 50% of the LD50 of methanol, ethanol, or isopropanol was employed by Traiger and Plaa (197 1) and Cantilena et al. (1979) whereas Cornish and Adefuin (1967) used 40% of the LD50 of methanol, t-butanol, and amyl alcohol. Ueng et al. (1983) treated the rats chronically with ethanol. One drawback of such studies is that secondary and tertiary mechanisms may interfere with the primary mechanism(s) of interactions. Moreover, the possibility of toxic interaction at low concentrations of the interactants cannot be reliably predicted from such studies. Systematic studies with a homologous series of alcohols have not been reported. Moreover, while most investigators have measured one or more parameters of liver injury, the issue of whether the alcohol-potentiated liver injury leads to increased lethality has not been investigated. In most studies, the degree of potentiation was evaluated by: (i) estimating elevation in plasma transaminases as an indication of liver injury; (ii) histopathological examination of liver and/or (iii) covalent binding of “CC14-‘4CHC13 metabolites to liver macromolecules. In the present investigation, hepatotoxicity was estimated by the leakage of cytoplasmic enzymes, aspartate and alanine aminotransferases (AST, ALT),3 into plasma, and by histopathological examination of liver sections by light microscopy. Lethality was measured as LD50 of Ccl4 or CHC13 after oral administration of the respective halomethane alone or after ’ Abbreviations AST, aspartate Schiff reagent.
used: ALT, alanine aminotransferase; aminotransferase; PAS, periodic acid-
MEHENDALE
prior exposure to methanol, ethanol, isopropanol, t-butanol, pentanol, hexanol, octanol, decanol, and eicosanol at a single, nontoxic dose administered orally. MATERIALS
AND
METHODS
Animals and treatment. Male Sprague-Dawley rats weighing 175-250 g were purchased from Charles River breeding laboratories (Willmington, MA) and maintained on Purina Chow rodent diet and water ad libitum for 5-9 days prior to the experiments. They were divided into four groups: (i) alcohol and halomethane vehicle control (i.e., saline or corn oil): (ii) alcohol control, IO mmol/kg (in corn oil or saline) + corn oil; (iii) CC4 or CHCIs alone: saline or corn oil + Ccl4 or CHC13 (0. I, 0.5, or 1 ml/kg), one of the doses was administered as I : I solution in corn oil; and (iv) alcohol and halomethane combination: alcohol IO mmol/kg + Ccl, or CHCI,. Alcohols and halomethanes were administered po. Eighteen hours before the dose of a single administration of Ccl,, CHC&, or corn oil vehicle, the animals received a given alcohol or a suitable vehicle such as corn oil or saline. The alcohol pretreatment regimen (10 mmol/kg, po) was chosen based on preliminary experiments which showed that none of the alcohols employed in this study caused liver injury upon a single oral administration to rats. Alcohols at this dose did not cause any elevation in plasma enzymes nor did they elicit any overt histological changes in the liver. The animals were returned to their respective cages after the administration of the chemicals. All animals were housed in plastic cages over untreated corncob bedding in our central facilities at 25 f 1°C and 50-800/u relative humidity. The following depicts the treatment protocol employed in these studies: I Saline/corn
oil
I Alcohol
oil
Saline/corn
518 hr Saline/corn
118 hr
I Saline/corn
oil
118 hr
118 hr
CC14 or CHCls
CC& or CHCI,
L24 hr
./24 hr
Oil
L24 hr
./24 hr
Plasma enzymes
I Alcohol
& histopathology
of Liver.
Plasma enq’mes. Twenty-four hours after the administration of Ccl,. CHC&, or corn oil, the animals were lightly anesthetized with diethyl ether, and blood was collected with heparinized syringes. Plasma was separated by centrifugation ofblood, and the extent of liver damage was assessed by estimating the activities of AST and ALT in the plasma. The enzymes were measured according to the method of Reitman and Frankel (1957) using a Sigma kit (procedure No. 505). Histopathology. When the animals were killed under ether anesthesia, the livers were rapidly excised, rinsed in
ALCOHOL-POTENTIATED
c23000 3 ~2000 E 4 “E1000 z e 64
0 : Saline/ . : Methanol A : Ethanol
corn
CC& AND CHCll TOXICITY
oil
I
A: lsopropanol
0 : Saline/
corn
r : Hem’01 v: 0ctano1
431 b
oil
,m.,.r.nnl 6 : D,,....,. 0 : Eicosanol
0 : t-Butanol n : Pentanol ,/I *ye/; ; 1/- i+=yzs*
om-h
fi/
n
0
0
0.1
0.5
1.0
CC14
CC14
(ml/kg)
(ml/kg)
FIG. I. Effects of various alcohol pretreatments on CC&-induced elevation of plasma AST. Male S-D rats received a single oral administration of the indicated alcohol (10 mmol/kg) in either saline or corn oil vehicle. Eighteen hours later, they received a single oral administration of a solution of CCL, (0.1,0.5, or 1 ml/kg) in corn oil, or corn oil alone. Twenty-four hours later, the plasma transaminases were determined. The figure shows plasma AST activities for control and treated animals. For the sake of clarity, two panels (a and b) are used to show the results. Means f SEM of three to five individual determinations. An asterisk indicates that the value is significantly different from the controls (p c 0.05). With the exception of eicosanol. all alcohols potentiate the CC&-induced elevation of AST. Significance: Response from 0.1 ml CC&/ kg is significantly different from I ml CC&/kg, but not from 0.5 ml CC&/kg; 0.1 ml CC&/kg is significantly different from 0.1 ml CCWkg in combination with methanol, ethanol, isopropanol, t-butanol, pentanol. hexanol, or octanol. Similarly, 0.5 ml CC&/kg alone is significantly different from various alcohols + 0.5 ml CC&/kg combinations except decanol and eicosanol. Also, I ml CC&/kg alone is significantly different from 1 ml CC&/kg in combination with various alcohols, except eicosanol.
0.9% NaCl, blotted, and weighed. A portion of each liver was sliced and fixed in phosphate-buffered 4% formaldehyde and then embedded in paraffin. Sections were stained with either periodic acid-Schiff (PAS) reagent or hematoxylin and eosin for histopathological evaluation under a light microscope.
0: .:
Saline/ Methanol
corn
oil
0: w:
t-Butanol Pentand
”
” 0.5 CC14
Lefhalitv. For 1Cday LD50 determination, four groups of rats consisting of four rats per group were treated po with the indicated alcohol (10 mmol/kg). Eighteen hours later, either CC4 or CHCls was administered in a geometric progression such that one group received one of the four doses. The number of dead ani-
1.0
(ml/W
rc
0
0.1
0.5
1.0
CC14 (ml/kg)
FIG. 2. Effects of various alcohol pretreatments on CC&-induced elevation of plasma ALT. Treatment and other details are as indicated in Fig. 1. Results are means + SEM of three to five individual determinations. Unless otherwise stated, all the values are significantly different from the respective controls (p G 0.05). (b) Eicosanol increased liver injury only at the I ml CCL/kg dose level. Significance: Response from 0.1 ml CC&/kg is significantly different from 1 ml CC&/kg, but not from 0.5 ml CC&/kg alone; 0.1 ml CC&/kg is significantly different from 0.1 ml CC&/kg in combination with methanol or isopropanol, tbutanol, pentanol, hexanol, or octanol. Similarly, 0.5 ml CC&/kg alone is significantly different from various alcohols + 0.5 ml CC&/kg combinations except ethanol, decanol, and eicosanol. Also, I ml CCL, atone is significantly different from 1 ml CC&/kg in combination with various alcohols.
432
RAY AND MEHENDALE
mals was recorded during two daily observations for 14 days, and the LD50 was determined according to the procedures of Thompson (1947) and Weil (I 952). Sixteen rats were employed for each of the alcohols used. For the LD50 determination of CCL, and CHCI, alone, the rats received corn oil vehicle 18 hr prior to the respective halomethane. Statistics. Statistical significance was determined by ANOVA followed by a Dunnett’s t test. Asterisks in the tables and figures indicate the statistical significance of the data points, and the levels of significance have been indicated in each table and figure legend. Significance of LD50 values was determined by 95% confidence limit intervals.
RESULTS 1. Changes in Plasma Enzymes Figure 1 shows the effects of various alcohol pretreatments on plasma AST levels. All alcohols employed except eicosanol increased plasma AST levels after the administration of Ccl.,. None of the alcohols alone increased AST levels significantly (data not shown). CC& alone, depending upon the dosage employed, increased AST levels significantly. However, the increase in AST levels in the alcohol + CC& group was severalfold higher than that with Ccl, alone, indicating a significant enhancement of liver injury. Among all the alcohols, t-butanol, methanol, and hexanol were more effective than the others in enhancing the Ccl,-induced serum transaminase elevations. Figure 2 shows the effects of various alcohol pretreatments on plasma ALT levels. The pattern of enhancement closely paralleled AST levels. With the exception of eicosanol, the rest of the alcohols increased ALT activity after the administration of CCL, indicating thereby that the combination treatment elicits severalfold greater toxicity when compared to either of the two agents individually. In this series, hexanol was found to be most potent, whereas methanol, isopropanol, t-butanol, and pentanol closely paralleled each other. Figure 3 shows the effect of various alcohol pretreatments on CHCl, liver injury as as-
sessed by plasma AST activity. All the alcohols increased serum AST levels after the administration of CHC& . However, the degree of increase is lower than that with the alcohol + CC& combination treatment. Eicosanol pretreatment did not result in any enhancement of CHCl,-induced plasma AST elevation. In contrast to the methanol + CCL, combination, the methanol + CHC13 combination was less effective in the elevation of the serum transaminases. Likewise, ethanol and isopropanol were weaker potentiators. Figure 4 shows the effect of various alcohol pretreatments on CHC& toxicity assessed by plasma ALT activity. It appears from the results that either alcohol or CHC13 when administered individually was incapable of increasing plasma ALT levels, whereas alcohol followed by a single dose of CHC& elicited significant liver injury as indicated by significantly elevated plasma ALT levels. However, eicosanol pretreatment failed to cause a CHC&-induced elevation of plasma ALT activity. Interestingly, decanol caused a greater potentiation of CHC13 toxicity in comparison to its effects on Ccl, toxicity. 2. Histopathology Liver sections were examined under a light microscope for the following signs of injury. Hepatocellular necrosis, swollen or ballooned cells, fatty infiltration and sinusoidal dilatation, inflammation, and infiltration by polymorphonuclear cells. Histopathological examination of the liver sections yielded findings which were consistent with the plasma transaminase data. Figure 5a shows the photomicrographs of PAS-stained liver sections from variously treated rats. Control (top left; saline + corn oil treatment): the photomicrograph shows normal architecture ofthe liver. CCL, (0.5 ml/ kg) elicited very limited centrilobular necrosis (top right) accompanied by findings typical of CCL, hepatotoxicity. Hexanol (hexanol + corn oil; bottom left) did not cause any sig-
ALCOHOL-POTENTIATED 0 : Saline/ 0 : Yethmol A : Ethanol
Corn
433
Ccl, AND CHC& TOXICITY
a
oil
; ?I I
l* A* A* 0 0.5
0.1
CHC13
1.0
0
0.1
(ml/kg)
0.5
CHC13
1.0
(ml/kg)
FIG. 3. Effects of various alcohol pretreatments on CHCl,-induced elevation of plasma AST. Male S-D rats received a single oral administration of the indicated alcohol (10 mmol/kg) in either saline or corn oil vehicle. Eighteen hours later, they received a single oral administration of 1:1 solution of CHC& (0.1,0.5, or 1 ml/kg) in corn oil or corn oil alone. Twenty-four hours later, the plasma transaminases were determined. The figure shows plasma AST activities for controls and treated animals. For clarity, two panels (a and b) are used to show the results. Results are means + SEM of three to five individual determinations. Unless otherwise stated, all the values are significantly different from the control (p G 0.05). With the exception of eicosanol, all alcohols potentiated the CHClr-induced elevation of AST. Significance: Response from 0.1 ml CHC&/kg is significantly different from 1 ml CHC&/kg, but not from 0.5 ml CHCIJ kg; 0.1 ml CHC13/kg alone is significantly different from 0.1 ml CHC& in combination with methanol, tbutanol, pentanol, hexanol, octanol, or decanol. Similarly, 0.5 ml CHClJkg alone is significantly different from various alcohols + 0.5 ml CHCls/kg combinations except ethanol and eicosanol. Also, 1 ml CHCls/kg alone is significantly different from all the alcohols in combination with 1 ml CHClJkg, except eicosanol.
nificant liver injury except marginal fatty infiltration and sinusoidal dilatation. The hexano1 + CC& (0.5 ml/kg) combination treatment (bottom right) caused massive hepatic
r;;1500 5 g
r a^^,.
0 : Saline/ corn 0: YethanoI A : Ethanol
injury with the following characteristics: (i) centrilobular necrosis, typical of massive CCL+ injury; (ii) ballooned hepatocytes in the centrilobular and midzonal areas; (iii) mas-
oil
A: Isopropanol t-Butan : Pentan
0 CHC13
(ml/kg)
0.1
0.5 CHC13 (ml/kg)
1.0
---
FIG. 4. Effects of various alcohol pretreatments on CHCl,-induced elevation of plasma ALT. Treatments and other details are as indicated in Fig. 3. Results are means ? SEM of three to five individual determinations. Unless otherwise stated, all of the values are significantly different from the respective controls (p G 0.05). Eicosanol did not potentiate liver injury. Significance: Response from 0.1 ml CHCls/kg is significantly different from 1 ml CHC&, but not from 0.5 ml CHCls/kg; 0.1 ml CHCl&g alone is significantly different from 0.1 ml CHClJkg in combination with methanol, isopropanol, t-butanol, hexanol, octanol, or decanol. Similarly, 0.5 ml CHCl&g alone is significantly different from all the alcohols in combination with 0.5 ml CHCls/kg, except ethanol and eicosanol. Also, 1 ml CHClJkg alone is significantly different from all the alcohols in combination with 1 ml CHClr/kg, except eicosanol.
434
RAY
AND
MEHENDALE
ALCOHOL-POTENTIATED
sive depletion of glycogen, (iv) increased infiltration of inflammatory cells; and (v) dead cell debri scattered over a significant portion of the hepatic lobule. All of these findings were typical of livers of all animals receiving the hexanol + CCL, combination treatment. The same qualitative morphological alterations were evident in hexanol-pretreated rats receiving CC& at a dose of either 0.1 or 1 ml/kg. The degree of the injury seen in these livers paralleled the Ccl, dose administered. The histopathological findings described above typify all of the alcohol + Ccl, combination treatments. Neither alcohol treatments alone nor CC& at 0.1 ml/kg alone elicited any significant effects. The most significant effects of treatment with alcohol alone were sinusoidal dilatation and lipid accumulation. The effects of hexanol (Fig. 5a, bottom left) represent the worst case in this regard. CCL, alone at 0.5 ml/kg caused greater injury typified by the histological findings associated with extensive hepatolobular necrosis, ballooned cells, fatty infiltration, and infiltration of polymorphonuclear cells. Hexanol potentiated CC& toxicity to the greatest extent in comparison to all of the other alcohols. Eicosanol did not increase the toxicity of CCL, at any of the three doses of the latter employed in this study. The other alcohols potentiated liver injury to varying degrees in the general order of hexanol > tbutanol > pentanol > octanol % isopropanol > decanol > methanol > ethanol, with respect to histopathological alterations in the liver (data not shown). Figure 5b shows the liver histopathological findings of the toxic interaction between octanol and chloroform. Treatment with either chloroform (0.5 ml/kg) or octanol(l0 mmol/ kg) alone did not show any significant liver
CC& AND CHQ
435
TOXICITY TABLE 1
EFFECT OFVARIOUSALCOHOISON CC& LETHALITY Alcohol
LD50
95% Confidence limits
Control Methanol Ethanol Isopropanol t-Butanol Pentanol Hexanol Octanol Decanol
4.50 2.78 3.36 2.99 1.06* 1.OP 0.75s 1.49* 3.00
3.10-6.48 2.02-3.84 2.38-4.75 2.01-4.47 0.65-I .06 1.06-1.06 0.46- 1.22 1.00-2.23 2.01-4.47
Note. The alcohols were administered po as a 25% solution in either corn oil or saline, I8 hr prior to the administration of CC&. The LD50 values were determined by the method of Thompson (1947) and Weil(1952) after a 14-day observation. An asterisk indicates that the value is significantly lower than the LD50 of Ccl, alone (p G 0.05).
injury except for some fatty infiltration in livers of rats receiving CHC13 alone. However, the octanol + CHC13 combination treatment caused massive damage identical to the hexano1 + CCL, treatment. Hepatic injury was evident as follows: (i) ballooned hepatocytes in the centrilobular and midzonal areas; (ii) massive depletion of glycogen; (iii) extensive necrotic cells in the centrilobular and midzonal areas; (iv) fatty infiltration throughout the lobule: and (v) infiltration of polymorphonuclear cells in areas of hepatocellular necrosis. The interaction of octanol + CHQ depicted in Fig. 5b represents the most severe interaction among all alcohols employed in this study. Eicosanol did not enhance the toxicity of CHC& at any dose of the halomethane. Other alcohols caused enhancement of liver injury
FIG. 5. (a) All fields show PAS-stained liver sections from male S-D rats (10 m. X90): (top left) control; (top right) CC4 (0.5 ml/kg); (bottom left) hexanol(l0 mmol/kg); and (bottom right) hexanol(l0 mmol/ kg) + CC& (0.5 ml/kg). Massive liver damage is evident in the liver section from hexanol + CC&-treated rats. (b) All fields show PAS-stained liver sections from male S-D rats (10 m, X90): (top left) control; (top right) CHCI, (0.5 ml/kg): (bottom left) octanol (10 mmol/kg); and (bottom right) octanol (IO mmol/kg) + CHC& (0.5 ml/kg). Massive damage is evident in the liver section from octanol + CHCl,-treated rats.
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to a varying degree. In general, the order ranking for the enhancement of CHC13 liver injury was as follows: hexanol > octanol > isopropanol L t-butanol > decanol > methanol > ethanol (data not shown).
3. Lethality Eflects of various alcohol pretreatments on the LDSO of CC&. These studies were conducted in order to determine if alcohol-potentiated CC& liver injury was of any consequence to animal survival. The findings (Table 1) reveal rather interesting observations in this regard. Although a number of alcohols are capable of potentiating hepatotoxicity, not all alcohols which potentiate hepatotoxic effects increased the lethal effects. The alcohols fell into three groups: the first group of alcohols, which potentiates liver injury of Ccl, but not CC4 lethality significantly, is represented by methanol, ethanol, isopropanol, and decanol. The second group of alcohols, which potentiates liver injury and also the lethal effects of CC& as evidenced by the decreased LD50 of CCL, comprises t-butanol, pentanol, hexanol, and decanol, t-butano1 being the most potent in this regard. The third group is represented by eicosanol, which potentiated neither the hepatotoxic nor the lethal effects of CC& (Table 1). Eflects of various alcohol pretreatments on the LD50 of CHClj. Table 2 shows the LD50 values of CHC13 and the alcohol + CHC13 treatment. The alcohol + CHC13 interaction resulted in greater lethality than the alcohol + CC& interaction. All of the alcohol pretreatments significantly decreased the LD50 of CHC13 in contrast to the alcohol + CC4 interaction. Octanol caused the maximal effects followed by t-butanol and isopropanol. Methanol caused the least decrease in the LD50 of CHCl3. On a comparative basis, the alcohol + CCL combination treatment did cause severe hepatotoxicity as indicated by the severalfold higher plasma transaminase levels. It appears
TABLE 2 EFFECT OF VARIOUS ALCOHOLS ON CHClr LETHALITY Alcohol
LD50
95% Confidence limits
Control Methanol Ethanol Isopropanol t-Butanol Pentanol Hexanol Octanol Decanol
1.99 0.84* 0.84* 0.501 0.50* 0.70* 0.63* 0.40* 0.80*
1.34-2.98 0.50-1.22 0.60-1.19 0.31-0.82 0.26-0.96 0.43-1.15 0.30-1.29 0.27-0.59 0.54-1.19
Note. The alcohols were administered po as a 25% solution in either corn oil or saline, 18 hr prior to the administration of CHCl,. The LD50 values were determined by the method of Thompson (1947) and Weil ( 1952) after a 14-day observation. An asterisk indicates that the value is significantly lower than the LD50 of CHC& alone (p c 0.05).
that the degree of potentiation alcohol + CHC13 combination and leads to greater lethality.
elicited by the is more potent
DISCUSSION Results from the present series of experiments provide some new and interesting information on alcohol-potentiated halomethane toxicity. In general, the alcohols fell into three categories in the case of alcohol + Ccl, interaction. However, the alcohols could be categorized into two groups in the case of alcohol + CHC13 interaction. These findings serve to illustrate that generalization from alcohol interaction studies with one halomethane may not be applicable to another halomethane. With the alcohol + Ccl4 interaction, group (i) consisting of methanol, ethanol, isopropanol, and decanol potentiated liver injury but did not affect animal survival significantly. Group (ii) consisting of t-butanol, pentanol, hexanol, and octanol potentiated liver injury and also significantly decreased animal sur-
ALCOHOL-POTENTIATED
vival, whereas group (iii) represented by eicosanol potentiated neither liver injury nor lethality. Results from the alcohol + CHC& interaction studies are somewhat different. Alcohols fell into two distinct categories: group (i) methanol, ethanol, isopropanol, t-butanol, pentanol, hexanol, octanol, and decanol potentiated liver injury and significantly affected animal survival, whereas group (ii) represented by eicosanol, failed to interfere with either of the two events, hepatotoxicity or lethality. The structure-activity relationship of alcohols to their potential for the toxic interaction with halomethanes is of some interest in predictive toxicology. It may be suggested from the present series of experiments that (i) the degree of potentiation is dependent on the number of carbon atoms on the straight chain alcohols and (ii) hepatotoxicity eventually leads to lethality during this toxic interaction at least with alcohols from 4 to 8 carbons in the case of CC& and from 1 to 10 carbons in the case of CHC&. No clear understanding of the mechanism of alcohol + CC& or alcohol + CHC& potentiation has developed (Lowery et al., 1981). The maximal potentiating effect is not seen in vivo until 18 hr after the administration of the alcohols (Traiger and Plaa, 197 I), when the alcohols would have dropped to undetectable levels in the blood. It was postulated that alcohols trigger a time-dependent series of events responsible for the potentiation of halomethane toxicity. The observation that isopropanol was ineffective in potentiating 14CC14binding when added directly to microsomes metabolizing CC4 (Sipes et al., 1973) is consistent with this concept. Various mechanisms have been proposed such as enhanced absorption in the intestine (Lamson et al., 1928) increased hepatomicrosomal cytochrome P450-mediated metabolism (Scholler et al., 1970; Ilett et al., 1973; Anders and Harris, 198 I), depletion of liver glutathione (Estler and Ammon, 1966; MacDonald, 1977), increased lipid peroxidation (Maling
CC&
AND
CHC&
TOXICITY
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et al., 1975), and a shift to hepatic hypermetabolism (Strubelt, 1978). The cascade of events during this toxic interaction and their precise role in the potentiation of liver injury remain elusive. The present series of experiments corroborate with several earlier reports: (i) Anders and Harris (1981) reported that 6.4 mmol isopropanol pretreatment did not affect the LD50 of Ccl4 significantly although metabolism was increased, and (ii) Reynolds et al. ( 1982) have reported a severalfold increase in CHC& metabolism after isopropanol pretreatment when compared to CC14. Our present findings that isopropanol potentiates hep atotoxic and lethal effects of CHC& and only potentiates hepatotoxic effects of CC& are consistent with these earlier reports. The marked ability of isopropanol to potentiate CC& hepatotoxicity has been demonstrated by various workers (Comish and Adefuin, 1967; Harris and Anders, 198 1; Ueng et al., 1983). Isopropanol appeared to enhance selectively the activity of one or more forms of cytochrome P450 which catalyze the oxidation of aniline and 7-ethoxycoumarin. The metabolism of ethyl morphine and benzo[a]pyrene was not affected by pretreatment with isopropanol. These results lend credence to the hypothesis that the cytochrome(s) affected by isopropanol may cause significant biotransformation of Ccl4 to an active metabolite, possibly phosgene, which causes a nonspecific destruction of cytochrome P450dependent monooxygenases (Ueng et al., 1983). While such mechanisms may offer an explanation for enhanced liver injury of CCL, upon isopropanol pretreatment, the reason for the lack of potentiated lethality is not apparent from such mechanisms. CHCl, is one of the metabolic intermediates of cytochrome P450-mediated CC& metabolism and is also known to be more toxic than Ccl,. It is well established that the hepatotoxic effects of CHC& are mediated by phosgene, a reactive intermediate metabolite of CHC13. Therefore, it would be reasonable to speculate that the mechanism by which a
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compound potentiates CHC13 hepatotoxicity might be dependent on either the increased phosgene production or diminished phosgene detoxification. Cianflone et al. (1980) have published the view that, above all, organelle susceptibility or other factors may play a significant role in the mechanism of potentiation. Purushotham et al. (1988) have identified one such factor in their recent studies. Mehendale et al. (1989) have proposed that an important determinant of the extent of liver injury is whether hepatocellular regeneration and hepatolobular restoration takes place. Thus, they are of the opinion that normally when limited liver injury occurs due to the widely accepted putative mechanisms upon the administration of a low dose of CHC13, the liver responds by stimulating hepatocellular regeneration and hepatolobular tissue repair (Mehendale et al., 1989; Mehendale, 1989a). Unless some event interferes with these restorative biological events, normally the animals are able to overcome such injury. A similar mechanism has also been advanced for CC& (Mehendale, 1989ab). In the present study, we have observed all alcohols with the exception of eicosanol to potentiate liver injury of CC& and CHC13. However, only a few of the alcohols potentiated lethal effects of Ccl, and most of them potentiated CHC13 lethality. Although speculative, it is tempting to rationalize these findings based on the following sequence. Pretreatment with alcohol increases the bioactivation systems for these halomethanes and initiation of liver injury occurs via the usual, widely accepted putative mechanisms. For those alcohols which did not increase the lethal effect of CC&, the liver tissue was able to respond by stimulating the tissue repair mechanisms, consisting of the stimulation of hepatocellular regeneration and hence hepatolobular restoration. At later time points, after most of the administered CC& has been exhaled, no additional injury occurs. With the tissue repair processes being stimulated, the limited injury is overcome in due time, as the newly divided cells replace the dead cells,
resulting in restoration of hepatolobular architecture, culminating in the complete recovery of the animals. Although these concepts will require experimental scrutiny and verification, our results with methanol, ethanol, isopropanol, and decanol in combination with CCL, are consistent with this sequence of events. A noteworthy point here is that higher doses of alcohols are required to potentiate CC& or CHC13 toxicity in comparison to the remarkable potentiation by chlordecone. Increased renal injury as a potential contributing factor for alcohol potentiation of CHC13 lethality (Hewitt et al., 1979) is a possible factor for consideration. We employed rats for our present studies. Renal toxicity of CHC13 has been reported in mice (Hewitt et al., 1979; Pohl, 1979; Zimmerman and Norbach. 1980; Smith and Hook, 1984). Potentiation of renal toxicity by CHC13 is not known to readily occur in the rat model (Kluwe, 198 1). Therefore, any significant contribution of renal toxicity in the present CHC13 lethality studies is unlikely. For the alcohols, which increased the lethality of Ccl, in addition to the liver injury, the same putative mechanisms are involved in initiating liver injury. However, in this case, the biological events leading to stimulation of hepatocellular division and tissue repair are impaired by mechanisms hitherto not well understood. The result is a progression of what is normally a recoverable liver injury leading to hepatic failure to a level inconsistent with survival. The extent of interference with the tissue repair processes is a function of dose response both with respect to the alcohol pretreatment and the halomethane treatment and also with respect to the specific halomethane. Thus, with CHC13 interaction, most of the alcohols cause a greater potentiation of liver injury and hence a greater potentiation of lethality. This is consistent with the known greater toxicity of CHC13 at comparable doses of CC4 and CHC13. These conceptual proposals are based on experimental evidence indicating that up to a limit, the degree of liver injury results in
ALCOHOL-POTENTIATED
the stimulation of a proportionate degree of tissue repair processes (Mehendale et al., 1989). Second, it has also been demonstrated that chemicals such as chlordecone, mirex, and phenobarbital differ in their potential for interfering with the stimulation of hepatocellular regeneration and tissue repair during interaction with Ccl, and CHC13 (Purushotham et al., 1988; Mehendale, 1989a,b, 1990; Mehendale et al., 1989).
CC&
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These studies were supported by a grant from the Air Force Office of Scientific Research (AFOSR-88-0009). Harihara M. Mehendale is the recipient of the 1988 Burroughs Wellcome Toxicology Scholar Award.
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