Developmental changes in rat liver alcohol dehydrogenase

Developmental changes in rat liver alcohol dehydrogenase

DEVELOPMENTAL BIOLOGY 105, 526-529 (1984) BRIEF NOTES Developmental Changes in Rat Liver Alcohol Dehydrogenase J. LAD,* WILLIAM J. SHOEMAKER,? AN...

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DEVELOPMENTAL

BIOLOGY

105, 526-529

(1984)

BRIEF NOTES Developmental

Changes in Rat Liver Alcohol Dehydrogenase J. LAD,* WILLIAM J. SHOEMAKER,? ANDHYAM

PUSHKARAJ *Department

of Medicine, Division Research Center,

of

Pharmacology, The Salk Institute Received

February

M-013

far

H, University of California, Biological Studies, P.O. Box

16, 1984;

accepted

in revised

L. LEFFERT*

San Diego, La Jolla, Cal&&a 92093, and tAlcoh 85800, San Diego, California 92108

form

May

22, 1984

Hepatic alcohol dehydrogenase activity and mass content change coordinately during development in male rats. Enzyme activity and mass content increase continuously after birth to 100 and 80% of maximal values within 6 weeks (2.6 + 0.4 pmole/min/g liver and 92 f 20 fig/g liver), respectively. When expressed per milligram of soluble proteins, both parameters peak at 3 weeks (0.052 + 0.002 rmole/min/mg protein and 2.0 f 0.4 pg/mg protein) and then decrease gradually to plateau levels. These decreases probably arise from a “surge” in soluble liver protein levels that occurs after weaning. Similar developmental patterns also occur in female rats. These findings are the first quantitative measurements of this enzyme in developing animals.

The developing animal is a useful model system in which to study hepatocyte gene expression (Greengard, 1977). An increase in alcohol dehydrogenase (alcohol:NAD+ oxidoreductase, EC l.l.l.l), an enzyme that plays a prominent role in controlling blood alcohol levels, has been seen during development in various mammals (Raiha et al, 1967; Smith et ah, 1971; Balak et al 1982). However, little is known about the molecular basis of this change-particularly whether or not the developmental increase in enzyme activity results from an increase in enzyme protein. In this report, using a new and sensitive assay to measure rat liver alcohol dehydrogenase mass content directly (Lad and Leffert, 1983b), we describe quantitative changes in rat liver alcohol dehydrogenase during development. These changes are compared to those occurring concomitantly in alcohol dehydrogenase activity, liver soluble proteins, and liver weight. MATERIALS

AND

METHODS

Animals. The following Sprague-Dawley rats were obtained from Zivic-Miller (Pittsburgh, Pa.): (i) timedpregnant females at 21 days gestation; (ii) mothers with 7- and 14-day old litters; (iii) males and females of ages 21,28,35,42, and 49 days; and (iv) approximately 23-week-old retired male breeders. Animals were housed at 21°C with 12-hr dark:light cycles and fed Purina rat chow and water ad libitum. Enzyme preparation. Animals were sacrificed by decapitation 7 days after arrival. Livers were removed, washed in buffer A (Tris-HCl, pH 8.0), blotted, weighed, and homogenized in 3 vol of buffer A at 4°C with a Bio-homogenizer (Biospec Products, Barttlesville, Okla.) for 15 set at full speed. Homogenates were centrifuged 0012-1606/S4 Copyright All rights

$3.00

0 1984 by Academic Press, Inc. of reproduction in any form reserved.

526

for 5 min at 10,OOOg and then for 50 min at 37,000g at 4°C. The supernatant volumes were measured and used to determine total soluble proteins, enzyme mass content, and enzyme activity. Routinely, greater than 95% enzymatic activity from the homogenates was recovered in supernatants. Enzyme assays. Alcohol dehydrogenase activity was measured as previously described (Lad and Leffert, 1983a). Activity was expressed as micromoles of NADH formed per minute (pmole/min). Enzyme mass content was quantitated by a new enzyme-linked immunoadsorbent assay (ELISA) developed in this laboratory (Lad and Leffert, 1983b). Pure alcohol dehydrogenase, used as the standard in these assays, was obtained from a preparation (stored as 70 pg enzyme/ml) that had been frozen at -20°C for 80 days. Soluble proteins were measured by a routine procedure (Lowry et aL, 1951) with bovine serum albumin (BSA) as the standard. Alcohol dehydrogenase isozymes were examined by isoelectric focusing as previously described (Lad and Leffert, 1983a). All values are expressed as the mean f SD. RESULTS

Livers at 18 days of gestation (gestation time 21 days) contained no assayable alcohol dehydrogenase activity. The earliest time at which fetal livers contained detectable alcohol dehydrogenase activity was found to be at 20 days of gestation (0.10 + 0.02 pmole/ min/g liver or 0.0026 f 0.0005 pmole/min/mg protein, n = 8). Figure 1A shows the hepatic alcohol dehydrogenase mass content levels from birth through adult life in male Sprague-Dawley rats. Total enzyme content (pg

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changes in male rat liver alcohol dehydrocontent (A); enzyme activity (B); and liver wet proteins (C). Total soluble proteins, enzyme mass content were determined as described Methods. Adult = retired breeders of about 24 activity is expressed as rmole/min (rmole Each point is the average of six determinations

enzyme/liver) increased continuously throughout life from 10.1 + 1.4 pg (n = 6) at birth to 86 -C 26 pg at 2 weeks, to 664 f 93 pg at 4 weeks, and then to 2780 + 330 pg at 24 weeks (adult). Greater rates of increases were seen after 3 weeks compared to earlier ages. These changes paralleled those observed for total enzyme activity (Fig. lB), total soluble proteins (Fig. lC), and wet weight (Fig. lC), which all increased continuously. By contrast, the specific mass ratio of enzyme to total soluble proteins increased rapidly at first, from low levels of 0.64 _t 0.06 pg/mg protein at birth to peak levels of 2.06 + 0.31 pg/mg protein at 3 weeks (Fig. 1A). This ratio then declined gradually to a plateau level of 1.15 kO.17 pg/mg protein at 8 weeks. This pattern paralleled the changes in specific activity of

527

NOTES

alcohol dehydrogenase, which also increased rapidly at first, from low levels of 0.012 + 0.002 pmole/min/mg at birth to peak levels of 0.052 +- 0.002 hmole/min/mg at 3 weeks (Fig. 1B). Enzyme specific activities decreased to levels of 0.038 + 0.002 pmole/min/mg at 5 weeks and levelled off at 0.033 + 0.005 pmole/min/mg at 24 weeks (Fig. 1B). The immunoassayable alcohol dehydrogenase levels reported here for adult rats are 9- to lo-fold lower than the previously observed values (Lad et al, 1983; Lad and Leffert, 1983b). This discrepancy arises from the use of frozen pure enzyme rather than unfrozen purified enzyme as the standard in the immunoassay. Long-term freezing apparently increases the number of antigenic sites exposed on the enzyme. Consequently, quantitative ELISA standard curves are shifted to the left with respect to the z axis (Fig. 2). Since 80-dayold frozen standard enzyme preparations were used here, the ranges of absolute values obtained (1.0-2.2 pg enzyme/mg protein) are lower than earlier ones (11-19 pg enzyme/mg protein, Lad et al, 1983). Regardless, the relative developmental differences should not be affected because all the samples in the current experiments were assayed simultaneously. Furthermore, identical values were obtained with fresh and once-frozen (for 6 days) homogenate samples. When the results from Fig. 1 are replotted to compare the developmental changes in alcohol dehydrogenase with respect to changes in total soluble proteins (by expressing the results as levels per gram wet weight of liver), the levels of alcohol dehydrogenase activity and mass content increase coordinately and plateau 5-6 weeks after birth (Fig. 3). By contrast, soluble proteins remained at constant levels of 39-44 mg/g liver for the first 3 weeks, increased rapidly to a level

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FIG. 2. ELISA standard curves using pure rat liver alcohol dehydrogenase stored under different conditions. Pure enzyme stored at 4°C for 4 days (Cl), at -20°C for 4 days (O), and at -20°C for 80 days (0) was used as the standard for quantitative immunoassay as described under Materials and Methods.

528

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FIG. 3. Developmental changes in total soluble proteins and alcohol dehydrogenase per gram of rat liver. The data are plotted from results in Fig. 1. Enzyme activity is expressed as rmole/min (rmole NADH formed/min).

of 71 + 3 mg/g liver at 6 weeks, and then increased gradually to a level of 80 f 5 mg/g liver at 24 weeks (Fig. 3). Since rat liver exhibits at least two forms of enzyme after isoelectric focusing (Lad and Leffert, 1983a), liver supernatants from 0 to 24 weeks were examined to see if the expression of these forms varied during development. All supernatants exhibited both forms of enzyme. There were no apparent changes in relative amounts of these forms during development (data not shown). In separate experiments, developmental changes in alcohol dehydrogenase levels in male and female Sprague-Dawley rats O-7 weeks of age were compared. Developmental patterns of enzyme activity and mass content were similar for both sexes (data not shown). However, at 7 weeks, female rats weighed less and had smaller livers than male rats. In addition, compared to their male counterparts, female livers contained lower levels of total soluble proteins and, consequently, lower levels of alcohol dehydrogenase activity and mass content (Table 1). However, the levels of specific enzyme mass content concentrations were comparable (131 f 23 and 113 + 23 pg enzyme/g liver in male and female rats, respectively). At 7 weeks, absolute specific enzyme activity (pmole/min/mg alcohol dehydrogenase) in females (19.8 + 3.0) was higher than in males (14.4 f 1.0). The reason for this difference is as yet unknown. However, this difference was not evident in any other age group. DISCUSSION

This communication describes coordinate changes in the levels of rat liver alcohol dehydrogenase activity

VOLUME

105. 1984

and mass content during development. The quantitative measurements of liver alcohol dehydrogenase mass content made here represent the first direct determinations of the amount of this liver enzyme during development. The findings are in agreement with in vitro results obtained with long-term primary cultures of adult rat hepatocytes which simulate developmental transitions in alcohol dehydrogenase (Lad et aL, 1982; Lad and Leffert, 1983b). The developmental patterns for alcohol dehydrogenase expression reported here fall into class “C,” as defined for developing liver enzymes by Greengard (1977). This class encompasses those differentiated functions whose activities emerge at or soon after birth. The molecular regulatory mechanism(s) underlying these changes remains unknown. The patterns and the ages at which adult enzyme levels are reached seem to change according to how the data are expressed. This is most apparent when activity or enzyme mass content levels are compared on the basis of per milligram protein or per gram liver wet weight. For example, peaks are seen 3 weeks after birth when the results are expressed per milligram of protein (Figs. 1A and B). By contrast, a gradual increase up to 6 weeks is seen when the same data are expressed per gram liver (Fig. 2). This may explain the variable observations in the developmental time courses of rat liver alcohol dehydrogenase activity reported earlier (Raiha et aL, 1967; Rawat, 1976; Sjoblom et ak, 1978). The results in Fig. 2 indicate that pure rat liver alcohol dehydrogenase upon long-term freezing undergoes subtle structural changes which are detectable by immunochemical methods. Therefore, the present studies do not rule out the possibility that such structural

HEPATIC

TABLE 1 ALCOHOL DEHYDROGENASE MASS CONTENT IN ~-WEEK-OLD MALE AND FEMALE RATS

LEVELS

Sex Parameter Whole body weight Wet liver weight Total soluble proteins Total enzyme activity Total enzyme mass content Enzyme mass content/ g liver

Units

Male 269

f

Female 20

214

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18

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14.6 * 2.2 987 i- 102

mg

1.89 i 0.24

1.32 + 0.17

I.&g

131 & 23

113 + 23

27.1

*

11.8 * 1.0 838 25.4

2.9

Note. The measurements were made as described and Methods. For all the parameters, the data are five determinations f SD. Differences between male were statistically significant (P < 0.05, Student’s parameters except “enzyme mass content/g liver,” significant differences were obtained.

f k

33 0.8

under Materials the averages of and female rats t test) for all in which case no

BRIEF

changes in the enzyme occur during development which lead to higher quantitation values by immunoassay. Since the process of sexual maturation in male rats begins at about 4 weeks, and because androgenic steroids like testosterone and dihydrotestosterone are reported to lower rat liver alcohol dehydrogenase (Rachamin et al., 1980; Cicero et ak, 1980; Mezey and Potter, 1982), it might be concluded that testosterone causes the decrease in the specific activity of alcohol dehydrogenase observed 3-4 weeks after birth. This explanation seems unlikely, however, for two reasons. First, the changes in enzyme mass content occur coordinately with changes in enzyme activity. Second, apart from differences (only at ‘7 weeks) in absolute enzyme specific activity (pmole/min/mg alcohol dehydrogenase), developmental patterns are essentially identical in male and female rats up to 7 weeks after birth. Alternatively, it seems more likely that the decreases in enzyme activity and mass content per milligram of protein which begin at 3-4 weeks result from a “surge” in the hepatic levels of total soluble proteins. This surge occurs at weaning (3 weeks of age) when the rats switch from the low-carbohydrate, highfat, and high-protein diet of the mother’s milk to a solid commercial diet containing 60-‘70% carbohydrate and low fat (Dymsza et ah, 1964). This work was supported by USPHS Grants (to H.L.L.) AA 03504, and AM 28215. Please address correspondence Leffert. We thank Sandy Dutky for typing the manuscript.

AM 28392, to H. L.

REFERENCES BALAK, K. J., KEITH, R. H., and FELDER, M. R. (1982). Genetic and developmental regulation of mouse liver alcohol dehydrogenase. J. Biol Chem. 257, 15,000-15,007. CICERO, T. J., BERNARD, J. D., and NEWMAN, K. (1980). Effect of

NOTES

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castration and chronic morphine administration on liver alcohol dehydrogenase and the metabolism of ethanol in the male SpragueDawley rat. J. PharmacoL Exp. Ther. 215, 317-324. DYMSZA, H. A., CZAJKA, D. M., and MILLER, S. A. (1964). Influence of artificial diet on weight gain and body composition of the neonatal rat. J. Nub. 84, 100-106. GREENGARD, 0. (197’7). Enzymic differentiation of human liver: Comparison with the rat model. Pediat. Res. 11, 669-679. LAD, P. J., HUBERT, J. J., SHOEMAKER, W. J., and LEFFERT, H. L. (1983). Comparison of liver alcohol dehydrogenases in Fischer-344 and Sprague-Dawley rats. Comp. B&hem. PhysioL 75B, 373-378. LAD, P. J., and LEFFERT, H. L. (I983a). Rat liver alcohol dehydrogenase. I. Purification and characterization. Anal. Biochem. 133, 350-361. LAD, P. J., and LEFFERT, H. L. (1983b). Rat liver alcohol dehydrogenase. II. Quantitative enzyme-linked immunoadsorbent assay. AnaL Biochem. 133, 362-372. LAD, P. J., SHIER, W. T., SKELLY, H., DE HEMPTINNE, B., and LEFFF,RT, H. L. (1982). Adult rat hepatocytes in primary culture. VI. Developmental changes in alcohol dehydrogenase activity and ethanol conversion during the growth cycle. Alcoholism: C&n. Exp. Res. 6, 64-71. LOWRY, 0. H., ROSEBROUGH, N. J., FARR, A. L. and RANDALL, R. J. (1951). Protein measurement with the Folin phenol reagent. J. BioL Chem. 193, 265-273. MEZEY, E., and POTTER, J. J. (1982). Effect of dihydrotestosterone on rat liver alcohol dehydrogenase activity. Hepatology 2, 359-365. RACHAMIN, G., MACDONALD, J. A., WAHID, S., CLAPP, J. J., KHANNA, J. M., and ISRAEL, Y. (1980). Modulation of alcohol dehydrogenase and ethanol metabolism by sex hormones in the spontaneously hypertensive rat. Biochem. J. 186,483-490. RAIHA, N. C. R., KOSKINEN, M., and PIKKARAINEN, P. (1967). Developmental changes in alcohol-dehydrogenase activity in rat and guinea-pig liver. B&hem J. 103, 623-626. RAWAT, A. K. (1976). Effect of maternal ethanol consumption on fetal hepatic metabolism in the rat. Ann. N. I: Acad Sci 273,175183. SJOBLOM, M., PILSTROM, L., and MORLAND, J. (1978). Activity of alcohol dehydrogenase and acetaldehyde dehydrogenase in the liver and placenta during the development of the rat. Enzyme 23, 108-115. SMITH, M., HOPKINSON, D. A., and HARRIS, H. (1971). Developmental changes and polymorphism in human alcohol dehydrogenase. Ann. Hum. Genet. (London) 34, 251-271.