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Age-related changes in hepatic microsomal mixed function oxidase in three strains of rats
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Pharmacological Research, Vol. 25, Supplement 2, 1992
Age-related changes in hepatic microsomal mixed function oxidase in three strains of rats . Annarita Meneguz and Hanna Michalek . Lab . Pharmacology, Istituto Superiore di Sanita, 00161 Roma .
Mixed function oxidase - Aging - Rat strains Hepatic endoplasmic reticulum, containing the mixed function oxidase system (MFO) is the major site of xenobiotic metabolism in mammals . There are several, but conflicting, reports on the changes of hepatic MFO in aging (for references see 1,2,3) . However, such investigations were performed on different strains of rats . On the other hand considerable strain-dependent differences in the responses of MFO to various treatments have been shown (4,5) . Recently strain-dependent differences in response to diisopropyl fluorophosphate have been reported from this laboratory (4) . The purpose of the present experiments was to assess whether age-related changes in MFO depend on strain . Therefore, hepatic microsomal aminopyrine N-demethylase, aniline hydroxylase and cytochrome P-450 levels were determined in 3- and 24-month Wistar, Fischer 344 and Sprague-Dawley male rats . The body weights (in g) of young and aged rats were the following : 290 + 6 and 721 + 10 for Wistar, 242 + 10 and 436 + 15 for Fischer 344, and 302 + 11 and 655 + 35 for Sprague-Dawley ; the respective liver weights (in g) were 9 .5 + 0 .6 and 11 .4 + 0 .7, 12 .2 + 0 .7 and 16 .2 + 1 .0, and 10 .3 + 1 .1 and 15 .7 + 1 .7 . Therefore the liver weights increased to a lesser extent than body weights, resulting in an age-related decline of liver/body weight ratio (of about 50% for Wistar and about 30% for Fischer 344 and Sprague Dawley rats) . The enzymatic assays were performed on 9,000 supernatants of 12 .5% homogenates prepared with 0 .01M phosphate buffer containing 1 .15% KCl, pH 7 .4 . Aminopyrine N-demethylase was measured by formation of formaldehyde, aniline hydroxylase as that of p-aminophenol, according to the methods of Mazel, as previously described (4) . The level of cytochrome P-450 was measured on microsome fraction obtained by 100 .000 g centrifugation of 9 .000 g supernatants and estimated by the carbon monoxide difference spectrum of dithionite reduced microsomes (4) . The microsomal proteins (in mg/g liver) did not differ substantially between various strains and/or ages (from 21 + 1 .2 to 25 + 1 .0) . The data on the enzyme and cytochrome P-450 levels (Table 1) show that the levels of aminopyrine N-demethylase and cytochrome P-450 in young rats were very similar in Wistar and Fischer 344 strains, for Sprague-Dawley they were lower by about 40% ; the levels of aniline hydroxylase were similar in rats of the three strains . The age-related changes differed considerably between strains, being the most pro-
1043-6618/92/25110283-02/$03 .00/0
© 1992 The Italian Pharmacological Society
284
Pharmacological Research, Vol. 25, Supplement 2, 1992
pounced for Fischer rats and the least pronounced for Wistar rats . In fact, factorial analysis of variance (age x strain) showed significant differences for age and strain with signi ficant interactions between strain and ache (F=17 .3 for amino pyrine N-demethylase, 25 .93 for aniline hydroxylase ani 24 .38 for cytochrome P-450, all p<0 .001) . Aminopyrine Ndemethylase, aniline hydroxylase and cytochrome P-450 levels were reduced by about 60% in aged Fischer rats and by 45% in Sprague -Dawley rats . On the other hand in Wistar rats there was only an about 20% reduction of aminopyrine N-demethylase . The overall data indicate that age-related changes in MFO activities are strain-dependent . They are in good agreement with the literature data (for references see 1-5), showing Fischer 344 rats as particularly susceptible both to aging and various treatments . Table 1 . Age-related changes in the hepatic mixed function oxydase system in three strains of rats . Strain
Age (months)
Aminopyrine N-demethylase
Aniline hydroxylase
Cytochrome P-450
WISTAR
3 24
238 + 18 196 + 24*
23 .0 + 4 .1 22 .3 T 3 .3
1 .13 + 0 .05 1 .23 + 0 .20
FISCHER 344
3 24
239 + 29 110 + 8**
25 .2 + 2 .6 9 .3 + 1 .0**
0 .99 + 0 .14 0 .45 + 0 .08**
SPRAGUE DAWLEY
3 24
150 + 10 96 + 13**
26 .2 + 2 .1 17 .5 + 0 .6**
0 .63 + 0 .06 0 .42 + 0 .03*
The data were obtained on 9,000 g supernatants . AminopyrineN-demethylase activity is expressed as mol of HCOH formed min/g of liver, that of aniline hydroxylase as nmol of p-aminophenol formed/min/g of liver . Cytochrome P-450 content is expressed in nmol/mg protein . Values represent mean + SE from six animals in each group . Post-hoc analysis between 3- and 24-month rats of each strain was assessed by Student's t-test : * p<0 .05, ** p<0 .01 . 1 . Rikans LE . Influence of aging on the susceptibility of rats to hepatotoxic injury . Toxicol . Appl . Pharmacol . 1984, 73 : 243-249 . 2 . Chengelis CP . Age- and sex-related changes in the components of the hepatic microsomal mixed function oxidase system in Sprague-Dawley rats . Xenobiotica 1988, 18 : 1211-24 . 3 . Van Geel CAJF, Van Bezooijen CFA . The effect of age on the oxidative metabolism of antipyrine by uninduced microsomal fractions of rat liver . Mech .Ageing Develop . 1990, 53 :169-77 . 4 . Meneguz A ., Michalek H . Effect of diisopropylfluorophosphate on hepatic microsomal systems in two strains of rats . Bull . Environ . Contam . Toxicol . 1990, 44 : 924-31 . 5 . Augustine JA, Zemaitis MA . A comparison of the effects of cyclosporine (CsA) on hepatic microsomal drug metabolism in three different strains of rat . Gen .Pharmac . 1989, 20 :137-41 .
Pharmacological Research, Vol. 25, Supplement 2, 1992
Age-related changes in hepatic microsomal mixed function oxidase in three strains of rats . Annarita Meneguz and Hanna Michalek . Lab . Pharmacology, Istituto Superiore di Sanita, 00161 Roma .
Mixed function oxidase - Aging - Rat strains Hepatic endoplasmic reticulum, containing the mixed function oxidase system (MFO) is the major site of xenobiotic metabolism in mammals . There are several, but conflicting, reports on the changes of hepatic MFO in aging (for references see 1,2,3) . However, such investigations were performed on different strains of rats . On the other hand considerable strain-dependent differences in the responses of MFO to various treatments have been shown (4,5) . Recently strain-dependent differences in response to diisopropyl fluorophosphate have been reported from this laboratory (4) . The purpose of the present experiments was to assess whether age-related changes in MFO depend on strain . Therefore, hepatic microsomal aminopyrine N-demethylase, aniline hydroxylase and cytochrome P-450 levels were determined in 3- and 24-month Wistar, Fischer 344 and Sprague-Dawley male rats . The body weights (in g) of young and aged rats were the following : 290 + 6 and 721 + 10 for Wistar, 242 + 10 and 436 + 15 for Fischer 344, and 302 + 11 and 655 + 35 for Sprague-Dawley ; the respective liver weights (in g) were 9 .5 + 0 .6 and 11 .4 + 0 .7, 12 .2 + 0 .7 and 16 .2 + 1 .0, and 10 .3 + 1 .1 and 15 .7 + 1 .7 . Therefore the liver weights increased to a lesser extent than body weights, resulting in an age-related decline of liver/body weight ratio (of about 50% for Wistar and about 30% for Fischer 344 and Sprague Dawley rats) . The enzymatic assays were performed on 9,000 supernatants of 12 .5% homogenates prepared with 0 .01M phosphate buffer containing 1 .15% KCl, pH 7 .4 . Aminopyrine N-demethylase was measured by formation of formaldehyde, aniline hydroxylase as that of p-aminophenol, according to the methods of Mazel, as previously described (4) . The level of cytochrome P-450 was measured on microsome fraction obtained by 100 .000 g centrifugation of 9 .000 g supernatants and estimated by the carbon monoxide difference spectrum of dithionite reduced microsomes (4) . The microsomal proteins (in mg/g liver) did not differ substantially between various strains and/or ages (from 21 + 1 .2 to 25 + 1 .0) . The data on the enzyme and cytochrome P-450 levels (Table 1) show that the levels of aminopyrine N-demethylase and cytochrome P-450 in young rats were very similar in Wistar and Fischer 344 strains, for Sprague-Dawley they were lower by about 40% ; the levels of aniline hydroxylase were similar in rats of the three strains . The age-related changes differed considerably between strains, being the most pro-
1043-6618/92/25110283-02/$03 .00/0
© 1992 The Italian Pharmacological Society
284
Pharmacological Research, Vol. 25, Supplement 2, 1992
pounced for Fischer rats and the least pronounced for Wistar rats . In fact, factorial analysis of variance (age x strain) showed significant differences for age and strain with signi ficant interactions between strain and ache (F=17 .3 for amino pyrine N-demethylase, 25 .93 for aniline hydroxylase ani 24 .38 for cytochrome P-450, all p<0 .001) . Aminopyrine Ndemethylase, aniline hydroxylase and cytochrome P-450 levels were reduced by about 60% in aged Fischer rats and by 45% in Sprague -Dawley rats . On the other hand in Wistar rats there was only an about 20% reduction of aminopyrine N-demethylase . The overall data indicate that age-related changes in MFO activities are strain-dependent . They are in good agreement with the literature data (for references see 1-5), showing Fischer 344 rats as particularly susceptible both to aging and various treatments . Table 1 . Age-related changes in the hepatic mixed function oxydase system in three strains of rats . Strain
Age (months)
Aminopyrine N-demethylase
Aniline hydroxylase
Cytochrome P-450
WISTAR
3 24
238 + 18 196 + 24*
23 .0 + 4 .1 22 .3 T 3 .3
1 .13 + 0 .05 1 .23 + 0 .20
FISCHER 344
3 24
239 + 29 110 + 8**
25 .2 + 2 .6 9 .3 + 1 .0**
0 .99 + 0 .14 0 .45 + 0 .08**
SPRAGUE DAWLEY
3 24
150 + 10 96 + 13**
26 .2 + 2 .1 17 .5 + 0 .6**
0 .63 + 0 .06 0 .42 + 0 .03*
The data were obtained on 9,000 g supernatants . AminopyrineN-demethylase activity is expressed as mol of HCOH formed min/g of liver, that of aniline hydroxylase as nmol of p-aminophenol formed/min/g of liver . Cytochrome P-450 content is expressed in nmol/mg protein . Values represent mean + SE from six animals in each group . Post-hoc analysis between 3- and 24-month rats of each strain was assessed by Student's t-test : * p<0 .05, ** p<0 .01 . 1 . Rikans LE . Influence of aging on the susceptibility of rats to hepatotoxic injury . Toxicol . Appl . Pharmacol . 1984, 73 : 243-249 . 2 . Chengelis CP . Age- and sex-related changes in the components of the hepatic microsomal mixed function oxidase system in Sprague-Dawley rats . Xenobiotica 1988, 18 : 1211-24 . 3 . Van Geel CAJF, Van Bezooijen CFA . The effect of age on the oxidative metabolism of antipyrine by uninduced microsomal fractions of rat liver . Mech .Ageing Develop . 1990, 53 :169-77 . 4 . Meneguz A ., Michalek H . Effect of diisopropylfluorophosphate on hepatic microsomal systems in two strains of rats . Bull . Environ . Contam . Toxicol . 1990, 44 : 924-31 . 5 . Augustine JA, Zemaitis MA . A comparison of the effects of cyclosporine (CsA) on hepatic microsomal drug metabolism in three different strains of rat . Gen .Pharmac . 1989, 20 :137-41 .