Carbon tetrachloride hepatotoxicity as a function of age in female Fischer 344 rats

ELSEVIER

Mechanisms of Ageing and Development 76 (1994) 89-99

Carbon tetrachloride hepatotoxicity as a function of age in female Fischer 344 rats Lora E. Rikans*, K. Roger Hornbrook, Yong Cai Department of Pharmacology, University of Oklahoma College of Medicine, Oklahoma City, OK 73190, USA Received 26 March 1994; revision received 20 June 1994; accepted 5 July 1994

Abstract

Severity of liver damage 24 h after intraperitoneal administration of carbon tetrachloride (0.2 mi/kg) was evaluated in female Fischer 344 rats aged 5, 14 and 28 months, i.e. in young adulthood, middle age and old age. Carbon tetrachloride-induced hepatotoxicity, as judged by the leakage of hepatic enzymes into the bloodstream and the disappearance of hepatic microsomal cytochrome P450, was much less severe in old rats than in young-adult rats. For example, serum sorbitol dehydrogenase (SDH) activity following carbon tetrachloride administration was 680 #mol/min/l in old rats compared with 1710 #mol/min/l in young-adult rats, and the loss of hepatic cytochrome P450 was 25% of the total amount in old rats compared with 50% of the total in young-adult rats. Spin trapping and electron spin resonance (ESR) spectroscopy were utilized to measure the conversion of carbon tetrachloride to trichloromethyl radicals in vivo. This primary bioactivation step occurred at similar rates in female rats aged 5, 14 and 28 months. In addition, the total nonheme iron contents in livers of rats in the three age groups were similar. Thus, the age associated attenuation of carbon tetrachloride-induced hepatotoxicity was not explained on the basis of decreased bioactivation to reactive species or decreased availability of iron for promotion of lipid peroxidation. The results suggest that other factors are important determinants of age-associated changes in sensitivity to toxic chemicals.

Keywords." Carbon tetrachloride; Trichloromethyl radical; Hepatotoxicity; Iron, Rat liver

* Corresponding author, College of Pharmacy, BMSB 753, OUHSC, POB 26901, Oklahoma City, OK 73190, USA. 004%6374/94/$07.00 © 1994 Elsevier Science Ireland Ltd. All rights reserved SSDI 0047-63 74(94)01483-3

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1. Introduction

The hepatotoxic actions of carbon tetrachloride have been investigated extensively [1-3]. It is generally agreed that bioactivation to reactive intermediates is required and that the primary step is reductive metabolism by the cytochrome P450 system to the trichloromethyl radical. The trichloromethyl radical is a reactive species that yields a variety of secondary metabolites, including the highly reactive trichloromethyl peroxy radical. Production of the latter occurs in an aerobic environment and results in the initiation of lipid peroxidation by hydrogen abstraction and homolytic cleavage. The trichloromethyl peroxy radical also dechlorinates, producing chlorine radical and phosgene, both of which are reactive species capable of covalent binding. The involvement of lipid peroxidation in carbon tetrachloride hepatotoxicity is widely accepted, although covalent binding may also contribute to liver damage. Cellular defenses protect against carbon tetrachloride hepatotoxicity by removing free radicals from the peroxidizing system. The most important defense mechanisms involve vitamin E and glutathione. In a previous study with male Fischer 344 rats, we found that the hepatotoxicity of carbon tetrachloride was unaffected by aging [4]. This finding was unexpected as male rats demonstrate a substantial age-related decline in hepatic cytochrome P450dependent metabolism along with a 45-65% decrease in covalent binding of 14Clabeled carbon tetrachloride to microsomal protein and lipid in vitro [5-8]. We hypothesized that the age associated reduction in carbon tetrachloride bioactivation was counterbalanced by diminished protection against oxidative damage, with a net result of no change in the extent of liver damage. The age-associated decline in hepatic microsomal cytochrome P450 is smaller in female Fischer 344 rats than in male rats (10-20% vs. 30-50%) [7,9]. Thus, in female rats, the balance might be shifted such that an age-associated increase in hepatotoxicity would occur. The purpose of the present study, therefore, was to determine if carbon tetrachloride-induced hepatotoxicity in female rats changed as a function of aging. The possible involvements of age-associated alterations in carbon tetrachloride bioactivation and hepatic iron content were also examined. 2. Materials and methods 2.1. Animals and treatment

Female Fischer 344 rats, obtained from the colony maintained for the National Institute on Aging by Harlan Industries (Indianapolis, IN), were acclimated to our facilities for at least 2 weeks before use. They were housed in pairs on hardwood bedding in a temperature- and humidity-controlled room with equal 12-h periods of light and dark. The rats were allowed free access to a standard laboratory ration (Purina) and water. At the time of the experiments, young-adult, middle-aged and old rats were 5, 14 and 28 months of age and weighed 201 + 3, 266 ± 6 and 278 ± 9 g, respectively. Old rats were excluded from the study if they were moribund or if their livers had tumors or visible evidence of leukemic infiltration; they were not excluded if they had cataracts or tumors in other parts of the body. Rats

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in the younger age groups had no apparent diseases. The rats received intraperitoneal (i.p.) injections of carbon tetrachloride (20% in corn oil) at a dose of 0.2 ml of carbon tetrachloride/kg of body weight. Control rats received an equal volume of corn oil. Food was not withdrawn before treatment because the effects of fasting on hepatotoxicity may be modified by the aging process.

2.2. Preparation of liver homogenates and microsomes Livers were homogenized in four parts of a solution containing 0.15 M KCI and 0.05 M Tris-HCl, pH 7.4. Samples of the homogenates were acidified immediately for determination of total nonheme iron. Microsomes were prepared by differential centrifugation as previously described [10]. The 100 000 x g supernatant fractions were frozen at -70°C and used for measurement of hepatic enzyme activities on the following day. 2.3. Hepatic enzyme activities Alanine aminotransferase (ALT), aspartate aminotransferase (AST), and sorbitol dehydrogenase (SDH) activities were determined in the 100 000 x g supernatant fractions of control rats. ALT and AST activities were measured by the RietmanFrankel method, using a diagnostic kit from Sigma Chemical Co. (St. Louis, MO); the activities are expressed in Sigma-Frankel (SF) units per mg protein. SDH activity was measured by a standard spectrophotometric procedure [11]; the activity is expressed as nmol/min/mg protein. Protein was estimated by a modification [12] of the Lowry procedure with bovine serum albumin as the standard. 2.4. Hepatotoxicity assessment Extent of liver damage was assessed on the basis of the leakage of hepatic enzymes into the bloodstream and the disappearance of liver microsomal cytochrome P450. Female rats aged 5, 14 and 28 months were euthanized 24 h after injections with carbon tetrachloride or corn oil. Blood was collected and centrifuged. Sera were stored frozen (-20°C) for analysis of serum ALT, AST and SDH activities on the following day. Livers were excised and weighed. Cytochrome P450 content in liver microsomes was determined from the reduced CO difference spectrum [13]. Serum ALT, AST, and SDH activities were determined using the procedures described above for measuring the activities in liver. The activities in serum are expressed as SF units per ml or/zmol per min per 1. 2.5. Trichloromethyl radical formation Ability to form trichloromethyl radical from CCI4 was measured in vivo using the spin trapping agent t-phenylbutylnitrone (PBN) [14-16]. Female rats of the three age groups were injected i.p. with a solution of PBN (20 mg/ml in water) at a dose of 100 mg PBN/kg of body weight. After 5 min, they were injected with carbon tetrachloride as described above. After an additional 10 min, the rats were decapitated and their livers removed. A 1- to 1.2-g portion of liver was homogenized with 2 ml of 0.05 M sodium phosphate buffer solution, pH 7.4, and 3 ml of toluene. The homogenates were transferred to centrifuge tubes with 3 ml of saturated sodium

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chloride solution and centrifuged at 40 000 x g for 20 min. Following removal of the toluene layer, the tube contents were resuspended and recentrifuged to increase recovery of the toluene fraction. The toluene extracts were stored at -70°C until used for electron spin resonance (ESR) spectroscopy. The toluene extracts (0.4 ml) were placed in quartz tubes and inserted in a chamber between the magnets of an IBM ER200D-SRC spectrometer. ESR spectra were obtained using microwave source power, 20 roW; modulation amplitude, 1.0 G; gain, 1 × 106; time constant, 163.84 ms; sweep time, 83.0 s; and 10 spectral accumulations. The system used for data accumulation has been described previously [17]. Signal intensities for the trichloromethyl adducts of PBN were compared by measuring the overall heights of the low field doublets (far left) and correcting to 1 g of liver.

2.6. Nonheme iron Total nonheme iron was measured in liver homogenates of control rats essentially as described by the International Committee for Standardization in Haematology for the measurement of serum iron in human blood [18,19]. In brief, 0.5 ml of homogenate was acidified with 0.5 ml of a solution containing 0.6 M trichloroacetic acid, 14 mM thioglycolic acid, and 1.0 mM HCI. The mixture was centrifuged and the supernatant decanted. The pellet was washed once with 1.0 ml of the precipitating solution and the supernatants combined. One-half ml of the combined supernatant solution was added to 0.5 ml of chromogen solution (1.5 M sodium acetate and 0.5 mM ferene). Absorbance was measured at 593 nm and compared with the absorbance of standard Fe(NH4)2(SO4)2 solutions. The measured recovery of iron from liver homogenates was 96 4- 3% (mean 4- S.E. of four experiments). 2. 7. Statistics The data were analyzed by one-way analysis of variance with Tukey's procedure for pairwise comparisons or by the rank sum test with the Mann-Whitney U statistic. The analyses were performed using a computerized statistical program (Statistix, Analytical Software, St. Paul, MN) and the level of significance was set at P < 0.05. 3. Results

3.1. Hepatic activities of enzymes used as markers for hepatotoxicity ALT, AST and SDH are hepatic enzymes that are released into the bloodstream when liver cells are damaged. We measured the activities of these enzymes in liver supernatant fractions of control female rats aged 5, 14 and 28 months, because it was useful to know the basal levels of hepatic activities in order to interpret differences in serum activities. The effect of aging on marker enzymes was most pronounced for hepatic ALT activity, which decreased 60% between young-adulthood and old age in female Fischer 344 rats (Table 1). Age-dependent decreases in AST and SDH activities were smaller. AST activity declined 33% between young-adulthood and middle age, and SDH activity declined 20% between young-adulthood and old age. As the protein contents of liver supernatant fractions from different age groups were similar, the results are the same when the activities are expressed per g of liver instead of per mg of protein.

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L.E. Rikans et al./ Mech. Ageing Dev. 76 (1994) 89-99 Table 1 Effect of aging on liver enzymesused as markers for hepatotoxicity Age(months)

ALT (SF unitamg)

AST(SF unitamg)

SDH(nmol/min/mg)

5 14 28

690±44 a 451 ± 18b 270 ± 52c

646± 22a 436 ± 47b 500± 49a'b

191 ± 11a 179 ± 3b 152 ± 8c

Enzyme activities were measured in 100 000 x g supernatant fractions of livers from control female Fischer 344 rats. Valuesare means ± SE for fiverats, expressed per mg of protein. Protein concentrations were 18.9 4- 1.0, 19.3 ± 0.7 and 19.2 ± 1.2 mg/ml. Liverweights for 5-, 14- and 28-month-oldrats were 3.1 ± 0.1, 3.0 ± 0.1 and 3.7 ± 0.3 g per 100 g of body weight, respectively. Means in vertical columns that do not share a commonsuperscript are significantly different, P < 0.05, analysis of variance.

3.2. Carbon tetrachloride hepatotoxicity Blood and liver samples were collected from female Fischer rats aged 5, 14, and 28 months, 24 h after injection with carbon tetrachloride or corn oil. Serum ALT, AST and SDH activities were elevated up to 350-fold in carbon tetrachloride-treated rats (Fig. 1). The serum enzyme activities resulting from carbon tetrachloride administration were 2.5-5 times lower in old rats than in young-adult rats. Serum SDH activity, which was the least affected by age-associated changes in liver levels, was 680 ~mol/min/1 in 28-month-old rats compared with 17 l0/zmol/min/1 in 5-month-old rats. In order to correct for age-associated changes in the amount of hepatic enzyme available for release by carbon tetrachloride treatment, the ratio of serum enzyme activity after carbon tetrachloride treatment to hepatic enzyme activity (determined in untreated rats) was calculated for each enzyme activity (Table 2). Given an average liver weight of 3.3 g/100 g of body weight and assuming an average plasma volume of 3.3 ml/100 g of body weight [29], the ratio of units/ml of serum to units/g liver approximates the fraction of the total activity that was present in the serum at the time of blood sampling. The ratios for old rats were less than half of the ratios for young-adult rats, indicating that the proportion of enzyme released by carbon tetrachloride treatment was substantially less in old age. Treatment with carbon tetrachloride also caused a decrease in the cytochrome P450 content of liver microsomes. The loss of cytochrome P450 was substantially smaller in old rats than in younger ones (25% vs. 50% of the total) (Fig. 2). Control rats (corn oil treated) demonstrated an age-associated decline in hepatic microsomal cytochrome P450 content, and the amount of decrease (21%) was similar to that reported previously [9]. Liver to body weight ratios were unchanged by treatment with carbon tetrachlor±de. The liver weights of carbon tetrachloride-treated vs. control rats (given as percent of body weight) were 3.2 4- 0.1 vs. 3.1 ± 0.1, 3.1 4- 0.1 vs. 3.0 4- 0. l, and 3.6 4- 0.5 vs. 3.7 4- 0.3 for rats aged 5, 14 and 28 months, respectively. 3.3. Metabolism o f carbon tetrachloride to trichloromethyl radical Female Fischer 344 rats aged 5, 14, and 28 months were injected with the spin

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L.E. Rikans et aL /Mech. Ageing Dev. 76 (1994) 89-99

4000 E

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Fig. 1. Effect of aging on carbon tetrachioride-induced hepatotoxicity in female rats. The leakage of hepatic enzymes into the bloodstream was determined 24 h after the administration of carbon tetrachloride in corn oil (0.2 ml CCl4/kg , i.p.) to female Fischer 344 rats aged 5, 14, and 28 months (dark bars). Control rats received an equal volume of corn oil (light bars). Values are means • S.E. for five rats. Means that do not share a common letter are significantly different, P < 0.05, rank sum test.

trapping agent PBN followed by the same dose of carbon tetrachloride as used for the hepatotoxicity experiments. The rats were killed after 10 min and their livers removed for measurement of ESR signals in toluene extracts. An ESR spectrum characteristic of the trichloromethyl radical adduct of PBN was obtained in samples

95

L.E. Rikans et al./ Mech. Ageing Dev. 76 (1994) 89-99 Table 2 Ratio of released enzyme activity to available enzyme activity Age (months)

ALT

AST

SDH

5 14 28

0.041 0.022 0.020

0.055 0.034 0.021

0.095 0.058 0.046

The ratios were calculated from data in Fig. 1 and Table l by dividing serum enzyme activities (in units or nmol/min per ml) after carbon tetrachloride treatment by hepatic enzyme activity (in units or nmol/min per g liver) for untreated rats. Units of enzyme activity/g liver = units/mg protein x nag protein/ml supernatant fraction x 5 ml supernatant fraction/g liver.

from carbon tetrachloride treated rats (Fig. 3). ESR signal heights varied considerably, and there were no significant differences between age groups (Table 3). The characteristic spectrum was absent from liver extracts of rats that were injected with PBN only. 3.4. Hepatic iron content T o t a l n o n h e m e i r o n was m e a s u r e d in w h o l e cell h o m o g e n a t e s o f livers f r o m c o n trol rats. T h e i r o n c o n t e n t s o f livers f r o m 5-, 14- a n d 2 8 - m o n t h - o l d f e m a l e rats w e r e similar ( T a b l e 4). T h e v a l u e s also w e r e similar to p r e v i o u s l y r e p o r t e d v a l u e s for y o u n g - a d u l t a n d m i d d l e - a g e d m a l e rats o f the s a m e strain [20].

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Fig. 2. Effect of aging on carbon tetrachloride-mediated disappearance of cytochrome P450 from liver microsomes of female rats. Hepatic microsomal cytochrome P450 was measured 24 h after the administration of carbon tetrachloride in corn oil (0.2 ml CCI4/kg, i.p.) to female Fischer 344 rats (dark bars). Control rats received an equal volume of corn oil (light bars). Values are means ± S.E. for five rats. Means that do not share a common letter are significantly different, P < 0.05, analysis of variance.

96

L.E. Rikans et a l . / Mech. Ageing Dev. 76 (1994) 89-99

i

3440

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[G] Fig. 3. ESR spectrum of the trichloromethyl adduct of PBN in a toluene extract of female rat liver. Liver samples were prepared for ESR analysis 10 min after the administration of carbon tetrachloride (0.2 ml/kg). The instrument conditions used in obtaining the spectrum are given in 'Materials and methods'. Table 3 Effect of aging on the metabolism of carbon tetrachloride to trichloromethyl radical Age(months)

N

ESR Signal Height (nun)

5 14 28

5 5 6

36 (0-85) 35 (0-57) 47 (25-85)

ESR signal intensities of the trichloromethyl radical adduct of PBN were measured in toluene extracts of livers from female Fischer 344 rats. The rats received the spin trapping agent PBN (100 mg/kg) followed by carbon tetrachloride (0.2 ml/kg) and their livers were removed 10 rain later. The instrument conditions are given in Methods section. Values are medians with ranges in parentheses. There are no significant differences between age groups. Table 4 Effect of aging on hepatic nonheme iron Age (months)

Nonheme iron (nmol/mg protein)

5 14 28

154-2 18 + 1 13 + 2

Total nonbeme iron content was measured in whole cell homogenates of livers from control female Fischer 344 rats. Values are means ± S.E. for five rats. There are no significant differences between age groups.

L.E. Rikans et al./ Mech. Ageing Dev. 76 (1994) 89-99

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4. Discussion Administration of a standard dose of carbon tetrachloride to young-adult, middleaged and old female Fischer 344 rats caused substantial leakage of hepatic enzymes into the bloodstream and destruction of hepatic microsomal cytochrome P450. Agedependent differences were observed in both of these indicators of hepatotoxicity, with the damage produced by carbon tetrachloride being about one-half as severe in livers of old rats as in livers of younger rats. These results do not support the widely-held concept that aging is accompanied by an increase in vulnerability to oxidative injury. It should be noted, however, that time courses for enzyme release after carbon tetrachloride administration were not determined for each age group, and it is possible that the apparent age-associated decrease in hepatotoxicity was the result of a delay or acceleration in development of liver damage. Age-associated changes in hepatic enzyme activities are common, and differences between age groups in serum enzyme activities following treatment with a hepatotoxicant might reflect changes in hepatic levels of activity rather than differences in the extent of hepatic injury. Our measurements of ALT, AST and SDH activities in livers of aging female Fischer 344 rats indicated that ALT was the most affected by the aging process and, as a consequence, the least useful for studies of aging effects on hepatotoxicity. Hepatic SDH was the least affected by aging. SDH is found almost exclusively in liver, and its presence in serum is a sensitive and specific marker for hepatic injury [21]. Thus, the 2.5-fold difference between young-adult and old rats in serum SDH activity following carbon tetrachloride treatment is indicative of a significant difference in liver damage. In any case, calculation of the fraction of hepatic enzyme released clearly demonstrated that the release of all three enzymes decreased substantially with aging (Table 2). Spin trapping and ESR spectroscopy were utilized to determine whether aging influenced the metabolism of carbon tetrachloride to the trichloromethyl radical. The spin trapping technique allows a short-lived free radical, such as the trichloromethyl radical, to react with a suitable chemical trap, such as PBN, to form a secondary, more persistent radical which can be characterized by ESR spectroscopy. Spin trapping experiments have shown conclusively that the trichloromethyl radical is formed during carbon tetrachloride metabolism in vivo [14,15]. The results of this study demonstrate that the metabolism of carbon tetrachloride to trichloromethyl radical in the intact female rat is unaffected by animal age. The age-associated decline in carbon tetrachloride hepatotoxicity, therefore, is not a result of decreased bioactivation. These results are consistent with previous findings in vitro showing that agedependent changes in the hepatic enzymes that metabolize carbon tetrachloride are minimal in female Fischer 344 rats [7,9]. The results are also consistent with previous reports of differences in carbon tetrachloride hepatotoxicity that occur without changes in bioactivation and suggest that other factors contribute to the amount of liver damage produced by the halocarbon [17,22,23]. Iron availability might be critical in determining the extent of liver damage from carbon tetrachloride. The role of iron in lipid peroxidation is well established. Moreover, the administration of iron markedly potentiates carbon tetrachloride

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L.E. Rikans et al./ Mech. Ageing Dev. 76 (1994) 89-99

hepatotoxicity in rats [24,25]. It was of interest, therefore, to determine the influence of aging on hepatic iron content. Our expectation, based on previous studies in male rodents [20,26], was that hepatic iron content would increase as a consequence of aging. Instead, we found that the total nonheme iron content in female rat liver was unchanged by aging. The age-dependent change in susceptibility to carbon tetrachloride-induced liver damage observed in this study obviously did not correlate with hepatic iron content. The cellular defenses that inactivate the toxic species generated by carbon tetrachloride metabolism are free radical scavenging systems involving vitamin E and glutathione [1-3]. It is important to mention, therefore, that vitamin E and glutathione concentrations in livers of female Fischer 344 rats do not change with age [27,28]. In summary, the results of this study demonstrate that the extent of liver injury in female rats 24 h after treatment with carbon tetrachloride is decreased substantially as a consequence of aging, whereas the metabolism of carbon tetrachloride to its reactive metabolite is unaffected by aging. These observations, together with our previous findings [4,6,27,28], suggest that factors other than bioactivation/inactivation (e.g. repair systems, hormonal influences, immune responsiveness) are critical determinants of age-associated changes in hepatotoxicity. Understanding the mechanisms that account for age-dependent alterations in sensitivity to toxic chemicals is an important goal.

Acknowledgements This work was supported in part by Grant No. AG04984 from the National Institute on Aging. The skilled technical assistance of Tony R. Lopez is gratefully acknowledged.

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