Toxicology Letters, 54 (1990) 183-188
183
Elsevier
TOXLET 02462
Effect of almond and anis oils on mouse liver alcohol dehydrogenase, aldehyde dehydrogenase and heart lactate dehydrogenase isoenzymes
F.S. Messiha Texas Tech University Health Sciences Center, School of Medicine, Lubbock, TX (U.S.A.)
(Received 12 June 1990) (Accepted 25 July 1990) Key words: Alcohol dehydrogenase; Aldehyde dehydrogenase; Almond oil; Anis oil
SUMMARY The effects of short-term intraperitoneal injection of diluted almond or anis oil on heart lactate dehydrogenase isoenzymes, liver alcohol dehydrogenase and subcellular aldehyde dehydrogenase were studied in the female mouse. Hepatic alcohol dehydrogenase was induced from control by administration of almond oil 3.2 g/kg/d for 7 days, or anis oil 1.6 g/kg/d for 7 days. Treatment with almond but not anis oil inhibited both cytoplasmic and mitochondrial liver aldehyde dehydrogenase. The mitochondrial isoenzyme with an apparently low K,,, was also inhibited by the almond oil trial. No significant changes occurred in heart lactate dehydrogenase isoenzymes by the treatments used. The enzymatic inhibition kinetics were found to be non-competitive. The apparent K,,, for almond-treated mouse aldehyde dehydrogenase was greater than the controls. This indicates lower substrate affinity for almond oil than for acetaldehyde. The results suggest adverse hepatic metabolic interaction between almond oil and alcohol.
INTRODUCTION
The volatile oils of almond (ALM) and anis (ANS) are widely used for flavoring of food, liqueurs and certain medical preparations. They are listed in several pharmacopeias and in USNP. They have also found applications as mild expectorants, aromatic carminatives for the relief of colic conditions and in the management of digestive flatulence. These oil extracts possess pharmacological properties due to certain biologically active ingredients. This is apparent for the ALM constituent amygdalin (Laetrile or vitamin BIT) and its chemotherapeutic potential, which remains to be Present address and correspondence:
Department of Pharmacology, University of North Dakota, School of Medicine, Grand Forks, ND 58203, U.S.A. 0378-4274/90/$3.50 @ 1990 Elsevier Science Publishers B.V. (Biomedical Division)
184
substantiated in man [l], and the insecticidal activity of some ANS components [2]. The aromatic odor and toxicity of ALM have been ascribed to its content of benzyl alcohol and the metabolic formation of hydrocyanic acid, from bitter ALM, respectively. The latter is formed by hydrolysis of amygdalin to sugar and mandelonitrile and subsequent metabolism of the latter to hydrogen cyanide and benzyl alcohol. The cyanide formed, which inhibits oxidative phosphorylation and cellular respiration, has also been implicated in the development of lactic acidosis and pulmonary edema [3]. This may affect lactate dehydrogenase (LDH) isoenzymes which are also affected by benzyl alcohol [4]. Moreover, the presence of these oils in liquoric alcoholic beverages may result in modulation of ethanol (ET) and acetaldehyde (AC) metabolizing enzymes since benzyl alcohol also alters their specific activities [5]. This provided the rationale for the present evaluation of the effects of ALM and ANS on mouse liver alcohol dehydrogenase (L-ADH), subcellular aldehyde dehydrogenase (L-ALDH) and cardiac LDH isoenzymes to determine the likelihood of such adverse metabolic interaction. METHODS
The subjects were adult female Sprague-Dawley mice (Sprague-Dawley Co., Madison, WI), 8-9 weeks of age. They were housed under a controlled 12-h light/dark cycle environment in groups of 3 per cage and had access to Purina pellet food and tap water ad libitum. The mice were randomly divided into 4 groups of 6 mice each and injected intraperitoneally once daily for 7 consecutive days with either saline 1.8 g/kg, ALM 3.2 g/kg, or ANS 1.6 g/kg. The alcoholic oil extracts (Macormic Co., Baltimore, MD) and a 95% ET stock solution were diluted with saline to obtain 18% ET concentration prior to injection. They were administered in equal volume, 0.1 ml/ 10 g body wt. Animals were sacrificed by decapitation and the Iivers and hearts were excised, blotted dry with filter paper and weighed after drainage of heart blood. Individual livers were homogenized in ice-cold 0.1 M KC1 (pH 6.8) buffer and were differentially centrifuged to isolate the subcellular mitochondrial (MT) and cytoplasmic components [6]. Aliquots of these were used for the determination of L-ADH [7], L-ALDH [8] and L-MT isoenzymes with apparently low (L) and high (H) &, [9]. The inhibition kinetics were assessed by the Lineweaver and Burk method [IO] and the protein determination was made by the biuret procedure. The individual hearts were likewise homogenized in the KC1 buffer. They were then centrifuged at 22000 xg for 90 min at 4°C and the supernatant was used for the determination of the LDH subunits H and M by a modified spectrophotometric assay [ 111. The results are expressed as means+SEM of specific activity, nmol/min/mg protein for L-ADH and L-ALDH or mmol/min/mg protein for H-LDH isoenzymes. Regression analysis was performed for the kinetic study and Student’s t-test was used for the statistical analysis.
18.5
RESULTS
Table I summarizes the effect of short-term administration of ALM or ANS on L-ADH, subcellular L-ALDH and H-LDH isoenzymes. Daily injection of ALM or ANS for 7 consecutive days induced mouse L-ADH from ET-control by approximately 27% (PC 0.01) and 26% (P
183
Elsevier
TOXLET 02462
Effect of almond and anis oils on mouse liver alcohol dehydrogenase, aldehyde dehydrogenase and heart lactate dehydrogenase isoenzymes
F.S. Messiha Texas Tech University Health Sciences Center, School of Medicine, Lubbock, TX (U.S.A.)
(Received 12 June 1990) (Accepted 25 July 1990) Key words: Alcohol dehydrogenase; Aldehyde dehydrogenase; Almond oil; Anis oil
SUMMARY The effects of short-term intraperitoneal injection of diluted almond or anis oil on heart lactate dehydrogenase isoenzymes, liver alcohol dehydrogenase and subcellular aldehyde dehydrogenase were studied in the female mouse. Hepatic alcohol dehydrogenase was induced from control by administration of almond oil 3.2 g/kg/d for 7 days, or anis oil 1.6 g/kg/d for 7 days. Treatment with almond but not anis oil inhibited both cytoplasmic and mitochondrial liver aldehyde dehydrogenase. The mitochondrial isoenzyme with an apparently low K,,, was also inhibited by the almond oil trial. No significant changes occurred in heart lactate dehydrogenase isoenzymes by the treatments used. The enzymatic inhibition kinetics were found to be non-competitive. The apparent K,,, for almond-treated mouse aldehyde dehydrogenase was greater than the controls. This indicates lower substrate affinity for almond oil than for acetaldehyde. The results suggest adverse hepatic metabolic interaction between almond oil and alcohol.
INTRODUCTION
The volatile oils of almond (ALM) and anis (ANS) are widely used for flavoring of food, liqueurs and certain medical preparations. They are listed in several pharmacopeias and in USNP. They have also found applications as mild expectorants, aromatic carminatives for the relief of colic conditions and in the management of digestive flatulence. These oil extracts possess pharmacological properties due to certain biologically active ingredients. This is apparent for the ALM constituent amygdalin (Laetrile or vitamin BIT) and its chemotherapeutic potential, which remains to be Present address and correspondence:
Department of Pharmacology, University of North Dakota, School of Medicine, Grand Forks, ND 58203, U.S.A. 0378-4274/90/$3.50 @ 1990 Elsevier Science Publishers B.V. (Biomedical Division)
184
substantiated in man [l], and the insecticidal activity of some ANS components [2]. The aromatic odor and toxicity of ALM have been ascribed to its content of benzyl alcohol and the metabolic formation of hydrocyanic acid, from bitter ALM, respectively. The latter is formed by hydrolysis of amygdalin to sugar and mandelonitrile and subsequent metabolism of the latter to hydrogen cyanide and benzyl alcohol. The cyanide formed, which inhibits oxidative phosphorylation and cellular respiration, has also been implicated in the development of lactic acidosis and pulmonary edema [3]. This may affect lactate dehydrogenase (LDH) isoenzymes which are also affected by benzyl alcohol [4]. Moreover, the presence of these oils in liquoric alcoholic beverages may result in modulation of ethanol (ET) and acetaldehyde (AC) metabolizing enzymes since benzyl alcohol also alters their specific activities [5]. This provided the rationale for the present evaluation of the effects of ALM and ANS on mouse liver alcohol dehydrogenase (L-ADH), subcellular aldehyde dehydrogenase (L-ALDH) and cardiac LDH isoenzymes to determine the likelihood of such adverse metabolic interaction. METHODS
The subjects were adult female Sprague-Dawley mice (Sprague-Dawley Co., Madison, WI), 8-9 weeks of age. They were housed under a controlled 12-h light/dark cycle environment in groups of 3 per cage and had access to Purina pellet food and tap water ad libitum. The mice were randomly divided into 4 groups of 6 mice each and injected intraperitoneally once daily for 7 consecutive days with either saline 1.8 g/kg, ALM 3.2 g/kg, or ANS 1.6 g/kg. The alcoholic oil extracts (Macormic Co., Baltimore, MD) and a 95% ET stock solution were diluted with saline to obtain 18% ET concentration prior to injection. They were administered in equal volume, 0.1 ml/ 10 g body wt. Animals were sacrificed by decapitation and the Iivers and hearts were excised, blotted dry with filter paper and weighed after drainage of heart blood. Individual livers were homogenized in ice-cold 0.1 M KC1 (pH 6.8) buffer and were differentially centrifuged to isolate the subcellular mitochondrial (MT) and cytoplasmic components [6]. Aliquots of these were used for the determination of L-ADH [7], L-ALDH [8] and L-MT isoenzymes with apparently low (L) and high (H) &, [9]. The inhibition kinetics were assessed by the Lineweaver and Burk method [IO] and the protein determination was made by the biuret procedure. The individual hearts were likewise homogenized in the KC1 buffer. They were then centrifuged at 22000 xg for 90 min at 4°C and the supernatant was used for the determination of the LDH subunits H and M by a modified spectrophotometric assay [ 111. The results are expressed as means+SEM of specific activity, nmol/min/mg protein for L-ADH and L-ALDH or mmol/min/mg protein for H-LDH isoenzymes. Regression analysis was performed for the kinetic study and Student’s t-test was used for the statistical analysis.
18.5
RESULTS
Table I summarizes the effect of short-term administration of ALM or ANS on L-ADH, subcellular L-ALDH and H-LDH isoenzymes. Daily injection of ALM or ANS for 7 consecutive days induced mouse L-ADH from ET-control by approximately 27% (PC 0.01) and 26% (P
