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NAD-linked mitochondrial aldehyde dehydrogenase in the rat testis
Gen. Ph.rm.~. Vol 12. pp 363 Io 364. 1981 Prinled in Greal Britain All rights rcr,er',cd
0306-3623 gl.090363-0210200,0 (_'opyrtghl ..1"31981 Pergamon Press Ltd
NAD-LINKED MITOCHONDRIAL ALDEHYDE DEHYDROGENASE IN THE RAT TESTIS F. S. MESSIHA Division of Toxicology, Department of Pathology and Psychopharmacology Laboratory, Department of Psychiatry. Texas Tech University Health Sciences Center School of Medicine, Lubbock, Texas. U.S.A. (Receired 10 February 1981) Abstract The rat testis possesses mitochondrial aldehyde dehydrogenase. The specific activity of the enzyme increases as a function of age and is induced by short-term intake of diluted ethanol solution.
INTRODUCTION
Chronic alcoholism is often associated with abnormal endocrine function in man (Gordon et al. 1976; Gerdes, 1978; Van Thiel & Lester, 1979; Wright, 1978). It is generally accepted that ethanol (ET) is metabolized sequentially to acetaldehyde (AC) and acetate and that ET toxicity may be mediated by AC. Thus, suggesting the importance of aldehyde dehydrogenase (EC 1.2.1.3), which catalyze the oxidation of AC to acetate in the underlying detoxification mechanism(s). Aldehyde dehydrogenase (ALDH) is distributed in various organs, is primarily a mitochondrial (MT) enzyme and is also present in the cytosol. An A L D H activity has been shown in the cytosolic fraction of the rat testis (Messiha & Girgis, 1978) and no information is available on testicular M T - A L D H . The present study reports on the presence of pyridine nucleotide-dependent M T - A L D H in the rat testis and investigates some of its properties.
sumed the preceeding day by the ET-drinking rats (pairfeeding). The results are expressed as means + SE of specific activity of ALDH and student's t test for independent means was used for the statistical analyses of the data. RESUI.TS
Figure 1 shows mean specific activity of hepatic and testicular A L D H present in the M T preparations (upper bar graph) and reciprocal plots of substrate concentration of acetaldehyde vs the velocity of the reaction. Mean testicular M T - A L D H was 4.9 + 0.5 nmol/min/mg protein compared to 25.2 _+ 6.7 units of the hepatic M T enzyme in the adult male rat. Figure I also illustrates the graphic presentation of the Lineweaver-Burk reciprocal plots showing approximately
,oL ,t,,
oat
.
METHODS
Male Sprague Dawley rats, were maintained on purina pellet food and water ad lihitum, unless otherwise specified. Experiments were performed on groups of adult male rats. 80 to 100 day old, and on sexually immature rats, 30 day old. Animals were kept in a room with automatic alternating 12h light and 12h dark cycles for at least one week prior to experiments. Animals were killed by decapitation and their testes and livers were removed, rinsed with 0.1 M KCI buffer, pH 6.8, blotted dry with filter paper, weighed and homogenized in the ice cold KCI buffer by glass homogenizer to obtain 10% (w/v) tissue homogenates. Testicular and liver homogenates were fractionated by differencial eentrifugation to obtain the MT preparation as the source of ALDH as described in details elsewhere (Messiha & Hughes, 1979). Biuret procedure was used for the protein determinations. The method of Lineweaver and Burk (1934) was used for determinations of V,,,,, and K,, of MT-ALDH The latter was assayed spectrophotometrically (Messiha, 1980) and the enzymatic activity is expressed as specific activity, nmol/min/mg protein, measured at 30' C. In a separate set of experiments, 10'~;i (w/v) ET solution was available as the sole drinking fluid for 10 or 30 consecutive days to group of rats maintained on purina pellet food. The latter was given in same daily quantity con363
-O,8
"O,08
OOG
04
0.8
Fig. 1. Double-reciprocal plot for the oxidation of acetaldehyde by mitochondrial preparations from rat hepatic and testicular tissues. The enzymatic activity was measured in 0.1 M sodium pyrophosphate buffer, pH 9.8, using constant concentration of NAD (0.5 raM) at 30~C. The concentration of acetaldehyde varied between 0.08 and 0.8 raM. Pyrazol was present in the assay mixture at 0.15 M concentration. Each point represents the mean +_ SE derive from five to seven independent determinations. The bar graph represents specific activity of aldehyde dehydrogenase (ALDH) measured in the liver and in the testis of the same group of rats. Number of determination is given between parenthesis.
364
F.S. MEssmA Table 1. The effect of forced drinking of 10% (w/v) ethanol (ET) solution on endogenous specific activity of mitochondrial testicular (T) aldehyde dehydrogenase (ALDH) in the rat
1.2
0.6
! -O.4
!
!
! -0.06
1
0.O8 I
!
I
!
I
0.4
!
I
!
!
!
0.8
/s
Fig. 2. Double-reciprocal plots for the oxidation of acetaldehyde by mitochondrial testicular preparations from young and adult rats. Experimental conditions for the enzymatic assay were identical to those listed under Fig. 1. Each point represents mean + SE of 4 independent determination derive from young 30 day old, or sexually mature, 81-90 day old. rats.
4.7 fold lower Km value of T-ALDH from the corresponding enzyme in the rat liver using acetaldehyde as the substrate. Figure 2 shows reciprocal plots of acetaldehyde concentrations as a function of the velocity of the ALDH reaction in the young~ 30 day old, animals and in the adult, 81-91 day old, rats. The V=,x was greater in the sexually mature rats than in the younger rats (P < 0.05) with both groups of rats possessing almost identical K= value. Table 1 shows the effect of drinking of 10o//o ET solution for 10 or 30 consecutive days on endogenous testicular MT-ALDH in the rat. Animals were pair fed and the 10o/o ET solution was the sole drinking fluid for the duration of time indicated. Continued intake of ET for 30 days resulted in induction of T-MT-ALDH from respective controls (P < 0.05). No changes in specific activity of this enzyme was noted subsequent 10 days of ET consumption. DISCUSSION The results demonstrate the presence of NADdependent MT-ALDH in the rat testis which is greater in the adult than in the sexually immature rat. This increase in ALDH was not associated with changes in the apparent Km indicating little changes in the a~nity of the enzyme towards the substrate acetaldehyde as a function of sexual maturity. The specific activity of ALDH in the testicular tissue is of moderate order and magnitude since it represents approximately 209/oof that present in the liver, the organ with the largest known source of activity for ALDH. However, testicular MT-ALDH possesses greater affinity towards acetaldehyde as a substrate compared to that of the hepatic enzyme. The intake of 109/o ET as the sole drinking fluid for 10 days, under controlled food consumption, exerted little effect on endogenous testicular MT-ALDH. However, prolonged ET drinking induced T-MTALDH from respective controls. This is analgeous to
Duration Drinking Fluid (Days)
ALDH T-weight (nMol/min/mg (g) protein) (n)
Water (Control) 10~o ET
30 30
3.4 + 0.2 3.4 + 1.3
2.3 + 0.1 3.9 +_0.4"
(6) (5)
Water 10~/~ET
10 10
3.0 + 0.2 2.8 _ 0.2
4.5 + 0.7 3.7 + 0.4
(6) (6)
Animals were 90-100 day old and were pair-fed throughout the duration of the experiment. * P < 0.05. ET-mediated metabolic effect on rat liver ALDH which depends on the ET dose and duration of ET administration. The foregoing observation on the presence of NAD-linked ALDH in the MT fraction of the rat testis in addition to that previously found in the cytosol (Messiha & Girgis, 1978) suggest a metabolic importance of ALDH on the gonads. Assessing the properties of this newly found enzyme may conceivably lead to a better understanding of certain aspects of the metabolic detoxification mechanism underlying ET-evoked responses on the endocrine gland. SUMMARY
The presence of mitochondrial NAD-dependent aldehyde dehydrogenase (ALDH) in the rat testis is reported. The V=,, of the testicular mitochondrial enzyme increases as a function of age without concomitant alteration in the apparent K,. Specific activity of testicular ALDH is approximately 20% of that present in the rat liver. Drinking of a 10°/0 ethanol solution for 30 consecutive days resulted in induction of ALDH in the rat testis. REFERENCES
GORDON G. G., ALTMANA., SOUTHEa~A., Ruan'~ E. & LIEBERC. (1976) Effect of alcohol administration on sex hormone metabolism in normal men. New Eru31. d. Med. 295, 793-797. GERDES H. (1978) Alkohol und Endokrinum. Internist 19, 89-96. LINEWEAVER H. & BURK D. (1934) Determination of enzyme dissociation constants, d. Am. Chem. Soc. 56, 568-666. MESSmA F. S. & GmG1S S. M. (1978) Aldehyde dehydrogenase activity in the male rat reproductive tissues. Proc. west. Pharmae. Soc. 21, 353-356. MLSSmA F. S. & HUGHESM. J. (1979) Liver alcohol and aldehyde dehydrogenase: Inhibition and potentiation by histamine agonists and antagonists. Clin. exp. Pharmac. Physiol. 6, 281-292. Mmsm^ F. S. (1980) Testicular and epididymal aldehyde dehydrogenase in rodents: Modulation by ethanol and disulfiram. Int. J. Andrology 3, 375-382. VA~ THmL D. H. & Lm~R R. (1979) The effect of chronic alcohol abuse on sexual function. Clin. endocr. Metab. 8, 499-510. WRIGHTJ. (1978) Endocrine effects of alcohol. Clin. endocr. Metab. 7, 351-373.
0306-3623 gl.090363-0210200,0 (_'opyrtghl ..1"31981 Pergamon Press Ltd
NAD-LINKED MITOCHONDRIAL ALDEHYDE DEHYDROGENASE IN THE RAT TESTIS F. S. MESSIHA Division of Toxicology, Department of Pathology and Psychopharmacology Laboratory, Department of Psychiatry. Texas Tech University Health Sciences Center School of Medicine, Lubbock, Texas. U.S.A. (Receired 10 February 1981) Abstract The rat testis possesses mitochondrial aldehyde dehydrogenase. The specific activity of the enzyme increases as a function of age and is induced by short-term intake of diluted ethanol solution.
INTRODUCTION
Chronic alcoholism is often associated with abnormal endocrine function in man (Gordon et al. 1976; Gerdes, 1978; Van Thiel & Lester, 1979; Wright, 1978). It is generally accepted that ethanol (ET) is metabolized sequentially to acetaldehyde (AC) and acetate and that ET toxicity may be mediated by AC. Thus, suggesting the importance of aldehyde dehydrogenase (EC 1.2.1.3), which catalyze the oxidation of AC to acetate in the underlying detoxification mechanism(s). Aldehyde dehydrogenase (ALDH) is distributed in various organs, is primarily a mitochondrial (MT) enzyme and is also present in the cytosol. An A L D H activity has been shown in the cytosolic fraction of the rat testis (Messiha & Girgis, 1978) and no information is available on testicular M T - A L D H . The present study reports on the presence of pyridine nucleotide-dependent M T - A L D H in the rat testis and investigates some of its properties.
sumed the preceeding day by the ET-drinking rats (pairfeeding). The results are expressed as means + SE of specific activity of ALDH and student's t test for independent means was used for the statistical analyses of the data. RESUI.TS
Figure 1 shows mean specific activity of hepatic and testicular A L D H present in the M T preparations (upper bar graph) and reciprocal plots of substrate concentration of acetaldehyde vs the velocity of the reaction. Mean testicular M T - A L D H was 4.9 + 0.5 nmol/min/mg protein compared to 25.2 _+ 6.7 units of the hepatic M T enzyme in the adult male rat. Figure I also illustrates the graphic presentation of the Lineweaver-Burk reciprocal plots showing approximately
,oL ,t,,
oat
.
METHODS
Male Sprague Dawley rats, were maintained on purina pellet food and water ad lihitum, unless otherwise specified. Experiments were performed on groups of adult male rats. 80 to 100 day old, and on sexually immature rats, 30 day old. Animals were kept in a room with automatic alternating 12h light and 12h dark cycles for at least one week prior to experiments. Animals were killed by decapitation and their testes and livers were removed, rinsed with 0.1 M KCI buffer, pH 6.8, blotted dry with filter paper, weighed and homogenized in the ice cold KCI buffer by glass homogenizer to obtain 10% (w/v) tissue homogenates. Testicular and liver homogenates were fractionated by differencial eentrifugation to obtain the MT preparation as the source of ALDH as described in details elsewhere (Messiha & Hughes, 1979). Biuret procedure was used for the protein determinations. The method of Lineweaver and Burk (1934) was used for determinations of V,,,,, and K,, of MT-ALDH The latter was assayed spectrophotometrically (Messiha, 1980) and the enzymatic activity is expressed as specific activity, nmol/min/mg protein, measured at 30' C. In a separate set of experiments, 10'~;i (w/v) ET solution was available as the sole drinking fluid for 10 or 30 consecutive days to group of rats maintained on purina pellet food. The latter was given in same daily quantity con363
-O,8
"O,08
OOG
04
0.8
Fig. 1. Double-reciprocal plot for the oxidation of acetaldehyde by mitochondrial preparations from rat hepatic and testicular tissues. The enzymatic activity was measured in 0.1 M sodium pyrophosphate buffer, pH 9.8, using constant concentration of NAD (0.5 raM) at 30~C. The concentration of acetaldehyde varied between 0.08 and 0.8 raM. Pyrazol was present in the assay mixture at 0.15 M concentration. Each point represents the mean +_ SE derive from five to seven independent determinations. The bar graph represents specific activity of aldehyde dehydrogenase (ALDH) measured in the liver and in the testis of the same group of rats. Number of determination is given between parenthesis.
364
F.S. MEssmA Table 1. The effect of forced drinking of 10% (w/v) ethanol (ET) solution on endogenous specific activity of mitochondrial testicular (T) aldehyde dehydrogenase (ALDH) in the rat
1.2
0.6
! -O.4
!
!
! -0.06
1
0.O8 I
!
I
!
I
0.4
!
I
!
!
!
0.8
/s
Fig. 2. Double-reciprocal plots for the oxidation of acetaldehyde by mitochondrial testicular preparations from young and adult rats. Experimental conditions for the enzymatic assay were identical to those listed under Fig. 1. Each point represents mean + SE of 4 independent determination derive from young 30 day old, or sexually mature, 81-90 day old. rats.
4.7 fold lower Km value of T-ALDH from the corresponding enzyme in the rat liver using acetaldehyde as the substrate. Figure 2 shows reciprocal plots of acetaldehyde concentrations as a function of the velocity of the ALDH reaction in the young~ 30 day old, animals and in the adult, 81-91 day old, rats. The V=,x was greater in the sexually mature rats than in the younger rats (P < 0.05) with both groups of rats possessing almost identical K= value. Table 1 shows the effect of drinking of 10o//o ET solution for 10 or 30 consecutive days on endogenous testicular MT-ALDH in the rat. Animals were pair fed and the 10o/o ET solution was the sole drinking fluid for the duration of time indicated. Continued intake of ET for 30 days resulted in induction of T-MT-ALDH from respective controls (P < 0.05). No changes in specific activity of this enzyme was noted subsequent 10 days of ET consumption. DISCUSSION The results demonstrate the presence of NADdependent MT-ALDH in the rat testis which is greater in the adult than in the sexually immature rat. This increase in ALDH was not associated with changes in the apparent Km indicating little changes in the a~nity of the enzyme towards the substrate acetaldehyde as a function of sexual maturity. The specific activity of ALDH in the testicular tissue is of moderate order and magnitude since it represents approximately 209/oof that present in the liver, the organ with the largest known source of activity for ALDH. However, testicular MT-ALDH possesses greater affinity towards acetaldehyde as a substrate compared to that of the hepatic enzyme. The intake of 109/o ET as the sole drinking fluid for 10 days, under controlled food consumption, exerted little effect on endogenous testicular MT-ALDH. However, prolonged ET drinking induced T-MTALDH from respective controls. This is analgeous to
Duration Drinking Fluid (Days)
ALDH T-weight (nMol/min/mg (g) protein) (n)
Water (Control) 10~o ET
30 30
3.4 + 0.2 3.4 + 1.3
2.3 + 0.1 3.9 +_0.4"
(6) (5)
Water 10~/~ET
10 10
3.0 + 0.2 2.8 _ 0.2
4.5 + 0.7 3.7 + 0.4
(6) (6)
Animals were 90-100 day old and were pair-fed throughout the duration of the experiment. * P < 0.05. ET-mediated metabolic effect on rat liver ALDH which depends on the ET dose and duration of ET administration. The foregoing observation on the presence of NAD-linked ALDH in the MT fraction of the rat testis in addition to that previously found in the cytosol (Messiha & Girgis, 1978) suggest a metabolic importance of ALDH on the gonads. Assessing the properties of this newly found enzyme may conceivably lead to a better understanding of certain aspects of the metabolic detoxification mechanism underlying ET-evoked responses on the endocrine gland. SUMMARY
The presence of mitochondrial NAD-dependent aldehyde dehydrogenase (ALDH) in the rat testis is reported. The V=,, of the testicular mitochondrial enzyme increases as a function of age without concomitant alteration in the apparent K,. Specific activity of testicular ALDH is approximately 20% of that present in the rat liver. Drinking of a 10°/0 ethanol solution for 30 consecutive days resulted in induction of ALDH in the rat testis. REFERENCES
GORDON G. G., ALTMANA., SOUTHEa~A., Ruan'~ E. & LIEBERC. (1976) Effect of alcohol administration on sex hormone metabolism in normal men. New Eru31. d. Med. 295, 793-797. GERDES H. (1978) Alkohol und Endokrinum. Internist 19, 89-96. LINEWEAVER H. & BURK D. (1934) Determination of enzyme dissociation constants, d. Am. Chem. Soc. 56, 568-666. MESSmA F. S. & GmG1S S. M. (1978) Aldehyde dehydrogenase activity in the male rat reproductive tissues. Proc. west. Pharmae. Soc. 21, 353-356. MLSSmA F. S. & HUGHESM. J. (1979) Liver alcohol and aldehyde dehydrogenase: Inhibition and potentiation by histamine agonists and antagonists. Clin. exp. Pharmac. Physiol. 6, 281-292. Mmsm^ F. S. (1980) Testicular and epididymal aldehyde dehydrogenase in rodents: Modulation by ethanol and disulfiram. Int. J. Andrology 3, 375-382. VA~ THmL D. H. & Lm~R R. (1979) The effect of chronic alcohol abuse on sexual function. Clin. endocr. Metab. 8, 499-510. WRIGHTJ. (1978) Endocrine effects of alcohol. Clin. endocr. Metab. 7, 351-373.