Fd Cosmer. Toxicol. Vol. IS. pp. 419-422. Pergamon Press 1911. Primed in Great Britam
INHIBITION
OF YEAST ALCOHOL DEHYDROGENASE BY DEHYDRORETRONECINE
P. S. SUN*,
M.-T. S. HsrA*t, F. S. CHU* and J. R. ALLEN*t
*Food Research Institure and Department of Food MicrobiologJl and Toxicology, tDeparrment of Pathology and Primate Research Center, Unioersity of Wisconsin, Madison. Wisconsin 53706. USA (Received
9 May
and
1977)
Abstract-The interaction of dehydroretronecine, a hepatocarcinogenic metabolite of the pyrrolizidine alkaloid monocrotaline, with yeast alcohol dehydrogenase (ADH), cysteine and bovine serum albumin (BSA) was studied. Dehydroretronecine inhibited ADH with an inhibition constant (KJ of 3.38 x lo-* M at pH 7.5 and 25°C. No inhibitory effect was observed when excess cysteine was added to the assay system. The interaction of dehydroretronecine with cysteine was demonstrated by spectrophotometric titration. Titration of dehydroretronecine alone at 240 nm showed an apparent pK value of 5.0. whereas titration of dehydroretronecine in the presence of excess cysteine resulted in an increase of pK from 5.0 to 8.5. Equilibrium dialysis data indicated appreciable binding of dehydroretronecine to BSA at pH 4.2 and 6°C.
INTRODUCTION
Plants containing the pyrrolizidine alkaloids are distributed worldwide, and include many species in a wide variety of genera including Senecio,Crotalaria, Heliotropium, Trichodesma and Amsinckia (Kingsbury, 1964). Livestock poisoning by consumption of pyrrolizidine alkaloid-containing plants has recently become a major problem in the Pacific northwest and other parts of the United States and the world (Deinzer, Thomson, Burgett & Isaacson, 1977). Consumption by man of foods and herbal medicines contaminated with pyrrolizidine alkaloids has resulted in acute hepatic veno-occlusivediseasewhich progresses to liver cirrhosis (McLean, 1970). In addition, animal experiments have shown that certain pyrrolizidine alkaloids are carcinogenic (Allen, Hsu & Carstens, 1975; Harris & Chen, 1970; Svoboda & Reddy, 1972), mutagenic (Cook & Holt, 1966; Culvenor, Downing, Edgar & Jago, 1969) and teratogenic (Green & Christie, 1961). Dehydroretronecine is the major detectable toxic metabolite of the pyrrolizidine alkaloid, monocrotaline (Hsu, Allen & Chesney, 1973). This pyrrole is responsible for ulceration and atrophy of the gastric mucosa, rhabdomyosarcomas (Allen et al. 1975) and megalohepatocytosis. The discovery of the oncogenic nature of dehydroretronecine prompted investigations of the pyrrole’s interaction with cellular macromolecules in uiuo and in vitro (Hsu, Robertson & Allen, 1976; Hsu, Robertson, Shumaker & Allen, 1975). Their data suggest a direct correlation of the levels of dehydroretronecine binding to cellular macromolecules with the lesions that deveiop in affected organs. Hayashi & Lalich (1968) have shown that injections of either mercaptoethylamine or cysteine will prolong survival and improve the growth rate of rats that receive monocrotaline parenterally. Recently, protective effects of dietary cysteine have been reported in rats affected by pyrrolizidine alkaloid poisoning (Buckmaster, Cheeke & Shull, 1976). This 419
paper presents an account ofthe binding of dehydroretronecine to cysteine and bovine serum albumin (BSA)and the inhibitory effect of dehydroretronecine on a thiol enzyme, yeast alcohol dehydrogenase (ADH). EXPERIMENTAL
Enzyme assay. ADH derived from yeast and nicotinamide adenine dinucleotide (NAD) were purchased from Sigma Chemical Co. (St. Louis, MO.). The activity of yeast ADH was determined by the method of Vallee & Hoch (1955) by measuring the rate of increasein absorbancy at 340 nm, 1 unit being defined as the amount of enzyme that reduced 1 pmol NAD/ min at 25°C under the conditions specified. ADH concentration was determined spectrophotometrically at 280 nm using an extinction coefficient, Ey&%, of 12.6 (Hayes & Valick, 1954) and a molecular weight of 151,000 (Kagi & Vallee, 1960). Inhibition studies. The inhibitory effect of dehydroretronecine on ADH activity was determined by analysis of the initial velocity of the enzyme-mediated reaction in the presence and absence of dehydroretronetine in the reaction mixture. All data were treated by the methods of Lineweaver & Burk (1934) and Hunter & Downs (1945). The ability of cysteine to block the inhibition was determined by adding freshly prepared cysteine solution to the reaction mixture containing dehydroretronecine, substrate and ADH. Spectral
analyses
and
spectrophotbmetric
titrations.
Absorption data were obtained by measuring the appropriate solutions in a Beckman Model DU spectrophotometer with a light path of 1 cm at room temperature. Spectrophotometric titrations were carried out in a Radiometer automatic titrator with a Radiometer Titrigraph type SBR2 and SBUl syringe burette as described by Chu (1971). Samples were withdrawn from the titration vessel at appropriate pH values, and optical density was measured at 24Omn.
P. S. SUN, M.-T.
420
Preparation of dehydroretronecine roretroneciue. Monocrotaline was seed of Crotalaria spectabilis by
and
S.
HSIA,
RESULTS of delrydroretronecine
on enzyme
activity
The inhibitory effect of dehydroretronecine on ADH activity is clearly demonstrated in Fig. 1, which also shows the effect of varying the concentration of dehydroretronecine on ADH activity at a constant concentration of substrate. To examine the type of inhibition produced, the substrate concentration was varied in the presence and absence of dehydroretronecine (7.84 x 10e5 M). The results, plotted according to the method of Lineweaver & Burk (1934) are shown in Fig. 2. The plot indicates that dehydroretronecine has a mixed-type (competitive and noncompetitive) effect on ADH. The inhibition constant (Ki) was found to be 3.38 x lo-’ M under the conditions studied. Blocking
effect
01 cysteine
on enzyme
inhibition
Table 1 shows the results of adding cysteine to the enzyme assay system at zero time in the presence of dehydroretronecine. The inhibitory effect of dehydroTable
1. Efict
of
cysteine
r
0.7 -
0.6 E E $ 0.5 m + 0 0.4 : z I) i 0.3 2 0.2 -
0.1 / 0
L
6 Reoct~on
time.
Fig. 1. ERect of dehydroretronecine on the enzyme activity The reaction was carried out in a pH 7.5 phosphate buffer at 25°C in the absence of dehydroretronecine (0) and in the presence of 7.84 x 10e5 M (0). 2.61 x 10m4~(@) and 3.27 x lo-“ M (m) DR. The conand NAD were centrations of enzyme, ethanol 3.05 x 10m6 M, 2.0 M and 2.5 x IO-” M, respectively. retronecine on ADH activity was blocked completely by the addition of cysteine at a concentration 8-15 times greater than that of dehydroretronecine. Spectrophotometric
titrations
Since dehydroretronecine showed the maximum difference between absorbance in acidic and basic conditions at 240 nm, spectrophotometric titrations were carried out at this wavelength. The titration of dehydroretronecine alone showed an apparent pK of 5.0 (estimated from the half-titration pH) as shown in Fig. 3 (curve d). In the presence of cysteine the pK value shifted from 5.0 to 8.5 (Fig. 3, curve b). Binding between dehydroretronecine and cysteine was evident from the drastic shift in position of the titration curve. of ADH
activity by
Relative enzyme activity (%)
+ cysteinet
100 100 75 100 62
+ cysteinet
100
*The assay conditions were the same as those described in Fig. 2. tFreshly prepared solution of cysteine was added at a final concentration of 3.3 x 10+&4.
fThe dehydroretronecine BThe dehydroretronecine
mln
of ADH.
on the inhibition dehydroretronecine
Incubation mixture* Control Cysteinet Dehydroretronecinej Dehydroretronecinel Dehydroretronecinfi Dehydroretronecine(i
and J. R. ALLEN 0.8
[3H]dehyd-
extracted from the the procedure of Koekemoer & Warren (1950). Dehydroretronecine was prepared from the parent alkaloid, monocrotaline, according to the method of Culvenor, Edgar, Smith & Tweeddale (1970). Dehydroretronecine tritirited at C-7 and C-9 (specific activity 20 &i/mg) was synthesized by a new procedure (M.-T. S. Hsia, unpublished data 1977) based on the reduction of I-formyl-7/?hydroxy-6,7-dihydro-SH-pyrrolizine with NaB3H, (New England Nuclear, Boston, Mass; > 100 mCi/ nmol). Equilibrium dialysis. Equilibrium dialysis was carried out at 6°C according to the method of Rosenberg & Klotz (1960). Detailed procedures were essentially those of Chu (1974) except that tritiated dehydroretronecine was used to calculate the free dehydroretronecine concentration. Radioactivity data were obtained by counting in 10ml of a solution of InstaGel universal liquid scintillation cocktail (Packard Instrument Co., Downers Grove, IL) using a Beckman Model 335 liquid scintillation spectrometer. A molecular weight of 65,000 (Spahr & Edsall, 1964) was used for the calculation of the molar concentration of BSA. Effect
F. S. CHU
concentration was 2.18 x lo-’ concentration was 4.36 x lob5
M. M.
ADH inhibition
by dehydroretronecine
I I/S.
,L
M
Fig. 2. Lineweaver-Burk plots for ADH; control reaction (01: reaction inhibited by 2.61 x 10mJ M dehydroretronecine (0). The reaction was carried out in a pH 7.5 phosphate buffer at 25°C. The concentrations of enzyme. ethanol and NAD were 1.52 x 10W6hl and 2.5 x lo-“M. respectively.
0.5
421
I 1.0 BSA
I
I
I
1.5 2.0 2.5 concentration.
I
I
3.0 3.5 10-4M
I 4.0
Fig. 4. Binding of [‘H]dehydroretronecine with BSA. The data were obtained by equilibrium dialysis at pH 4.2 and 6°C. The C3H]dehydroretronecine concentration was 1.13 x lo-*M(: the BSA concentration varied from 7.6 x 1O-6 to 3.85 x IO-‘M. there was no binding of BSA with dehydroretronecine at pH 7.0.
The binding curve of dehydroretronecine with BSA obtained from equilibrium dialysis in pH 4.2 citratephosphate buffer is shown in Fig. 4. The extrapolated value obtained from the curve indicates that 0.25 mol dehydroretronecine is bound by 1 mol BSA. However, 0.7 l--
Cd)
+ o 0.4-
2
4
6
8
IO
12
PH
Fig. 3. Spectrophotometric titration of cysteine and the dehydroretronecine-cysteine complex: (a) titration data of cysteine alone; (b) titration data of dehydroretronecinecysteine complex; (c) the difference between the data of curves b and a (b - a); (d) titration of free dehydroretronecine. The starting concentration of dehydroretronecine was 3.27 x 10Y4~.
DISCUSSION The inhibition of ADH activity by dehydroretronecine and the binding ofdehydroretronecine with cysteine and BSA have been demonstrated by spectrophotometric analyses and equilibrium dialysis. Inhibition of ADH by p-chloromercuribenzoate has been reported by Snodgrass, Vallee & Hoch (1960), and the inhibition was reversible on addition of excess glutathione. It is possible that the inhibitory effect of dehydroretronecine on ADH was due to the reaction of dehydroretronecine with the thiol groups in the active centre of ADH. The importance of the interaction with thiol compounds to the in ciao toxic action of pyrrolizidine alkaloids can be seen from the protective effect of ip-injected mercaptoethylamine and cysteine against monocrotaline intoxication in rats (Hayashi & Lalich, 1968). This is substantiated by the recent report that dietary cysteine has a protective effect on rats affected by pyrrolizidine alkaloid poisoning (Buckmaster, Cheeke 8c Shull, 1976). However, the protective mechanism of thiol compounds against pyrrolizidine alkaloid intoxication is obscure. Cysteine administration reduced the incidence of chromosomal breakage in Allium roots treated with monocrotaline or allied pyrrolizidine alkaloids (Avanzi, 1961). By analogy with data from radiobiology, it was suggested that cysteine protection against pyrrolizidine alkaloids was operating by lowering the intracellular oxygen tension. As far as we are aware, the present paper contains the first report on the inhibitory effect of a reactive pyrrolizidine alkaloid metabolite, dehydroretronecine, on the activity of an enzyme (ADH) under in vitro conditions, and the reverse effect of cysteine on such enzyme activity. Current literature shows that the alkylation of various cell components is one probable mechanism by which dehydroretronecine produces pathological alterations, whether they be gastric ulceration, hepatocellular necrosis or rhabdomyosar-
422
P. S. SUN, M.-T. S. HSIA, F. S. CHIJ and J. R. ALLEN
comas. On the basis of our present results, it might be postulated that thiol compounds exert their protective effect by competing with the nucleophilic components of the cells as potential target(s) for alkylation by the pyrrole metabolite(s). The interaction of dehydroretronecine with,. BSA was first studied by spectrophotometric analysrs (P. Sun and F. S. Chu, unpublished data 1976). Positive absorptivity was observed at 22&300nm when dehydroretronecine was incubated with BSA at pH 2.90, 5.22 or 7.20. However, calf-thymus DNA showed negative absorptivity under the same conditions. These results correlated with previous observations (Culvenor et al. 1969; Hsu et al. 1975) that there was a preferential binding of dehydroretronecine to proteins over other cellular components including nucleic acids. Results from equilibrium dialysis indicated no binding to BSA at pH 7.0. However, dehydroretronecine readily bound to BSA at pH 42. The most likely reason for this increased binding at a lower pH is increased protonation of the allylic hydroxyl group(s) in dehydroretronecine with subsequent dehydration, forming a relatively stable allylic carbonium ion which can undergo electrophilic alkylation of macromolecules. Although the significance of the interaction of dehydroretronecine with BSA at neutral pH is not known at present, BSA may serve as a transport agent for this pyrrole, thus providing a constant liberation of the toxic metabolite from the pyrrole-albumin complex to target tissue(s). Acknowledgements-This investigation, published as Primate Center Publication No. 17421, was supported in part by U.S. Public Health Service grants CA-13288, HL-10941 and RR-00167 from the National Institutes of Health.
REFERENCES Allen, J. R., Hsu, I. C. & Carstens, L. A. (1975). Dehydroretronecine induced rhabdomyosarcomas in rats. Cancer Res. 35, 997.
Avanzi. S. (1961). Chromosome breakage by pyrrolizidine alkaloids and modification of the effect by cysteine. Caryologia 14, 251. Buckmaster, G. W., Cheeke, P. R. & Shull, L. R. (1976). Pyrrolizidine alkaloid poisoning in rats: protective effects of dietary cysteine. J. Anim. Sci. 43, 464. Chu, F. S. (1971). Interaction of ochratoxin A with bovine serum albumin. Archs Biochem. Biophys. 147, 359. Chu, F. S. (1974). A comparative study of the interaction of ochratoxins with bovine serum albumin. Biochem. Pharmac. 23, 1105. Cook, L. M. & Holt, A. C. E. (1966). Mutagenic activity in Drosophila of two pyrrolizidine alkaloids. J. Genet: 59, 273.
Culvenor, C. C. J., Downing, D. T., Edgar, J. A. & Jago, M. V. (1969). Pyrrolizidine alkaloids as alkylating and antimitotic agents. Ann. N.Z Acad. Sci. 163, 837.
Culvenor, C. C. J.. Edgar, J. A., Smith. L. W. & Tweeddale, H. J. (1970). Dihydropyrrolizines. III. Preparation and reactions of derivatives related to pyrrolizidine alkaloids. Aust. J. Chem. 23, 1853. Deinzer, M. L., Thomson, P. A., Burgett, D. M. & Isaacson, D. L. (1977). Pyrrolizidine alkaloids: their occurrence in honey from tansy ragwort (Senecio jacobaea L.). Science, N.K-195, 497. Green. C. R. & Christie. G. S. f1961). Malformations in foe&l rats induced by ‘the pyrrolizidine alkaloid, heliotrine. Br. .I. exp. Path. 42, 369. Harris P. N. & Chen. K. K. (1970). Development of hepatic tumors in rats following ingestion of Senecio longilobus. Cancer Res. 30, 2881. Hayashi, Y. & Lalich, J. J. (1968). Protective effect of mercaptoethylamine and cysteine against monocrotaline intoxication in rats. Toxic. appl. Pharmac. 12, 36. Hayes, J. E. & Valick, S. F. (1954). Yeast alcohol dehydrogenase: molecular weight, coenzyme binding, and reaction equilibria. J. biol. Chem. 207, 225. Hsu, I. C., Allen, J. R. & Chesney, C. F. (1973). Identification and toxicological effects of dehydroretronecine, a metabolite of monocrotaline. Proc. Sot. exp. Biol. Med. 144, 835.
Hsu, I. C., Robertson, K. A., Shumaker, R. C. & Allen, J. R. (1975). Binding of tritiated dehydroretronecine to macromolecules. Res. Commun. them. Path. Pharmac. 11, 99.
Hsu, I. C., Robertson, K. A. & Allen, J. R. (1976). Tissue distribution, binding properties and lesions produced by dehydroretronecine in the nonhuman primate. ChemicoBiol.
Interactions
12, 19.
Hunter, A. & Downs, C. E. (1945). The inhibition of arginase by amino acids. J. biol. Chem. 157, 427. Kagiy J. H. R. & Vallee, B. L. (1960). The role of zinc in alcohol dehydrogenase. V. The effect of metal-binding agents on the structure of the yeast alcohol dehydrogenase molecule. J. biol. Chem. 235, 3188. Kinasburv. J. M. (1964). Poisonous Plants of the United Sites ind Canaria. p.‘425. Prentice-Hall, Inc., New Jersey. Koekemoer, M. J. & Warren, F. L. (1950). The Senecio alkaloids. VIII. The occurrence and preparation of the N-oxides. An improved method of extraction of the Senecio alkaloids. J. them. Sot. p. 60. Lineweaver, H. & Burk, D. (1934). The determination of enzyme dissociation constants. J. Am. them. Sot. 56,658. McLean, E. K. (1970). The toxic actions of pyrrolizidine (Senecio) alkaloids. Pharmac. Rev. 22, 429. Rosenberg, R. M. & Klotz, I. M. (1960). Dye binding methods. In A Laboratory Manual of Analytical Methods of Protein Chemistry. Vol. 2. Edited by P. Alexander and R. J. Block, p. 133. Pergamon Press, Oxford. Snodgrass, P. J., Vallee, B. L. & Hoch, F. L. (1960). Effects of silver and mercurials on yeast alcohol dehydrogenase. J. biol.
Chem.
235, 504.
Spahr, P. F. & Edsall, J. T. (1964). Amino acid composition of human and bovine serum albumin mercaptoalbumins. J. biol. Chem.
239, 850.
Svoboda, D. & Reddy, J. K. (1972). Malignant tumors in rats given lasiocarpine. Cancer des. 32,908. Vallee, B. L. & Hoch, F. L. (1955). Zinc, a component of yeast alcohol dehydrogenase. Proc. natn. Acad. Sci. U.S.A.
41, 327.
INHIBITION
OF YEAST ALCOHOL DEHYDROGENASE BY DEHYDRORETRONECINE
P. S. SUN*,
M.-T. S. HsrA*t, F. S. CHU* and J. R. ALLEN*t
*Food Research Institure and Department of Food MicrobiologJl and Toxicology, tDeparrment of Pathology and Primate Research Center, Unioersity of Wisconsin, Madison. Wisconsin 53706. USA (Received
9 May
and
1977)
Abstract-The interaction of dehydroretronecine, a hepatocarcinogenic metabolite of the pyrrolizidine alkaloid monocrotaline, with yeast alcohol dehydrogenase (ADH), cysteine and bovine serum albumin (BSA) was studied. Dehydroretronecine inhibited ADH with an inhibition constant (KJ of 3.38 x lo-* M at pH 7.5 and 25°C. No inhibitory effect was observed when excess cysteine was added to the assay system. The interaction of dehydroretronecine with cysteine was demonstrated by spectrophotometric titration. Titration of dehydroretronecine alone at 240 nm showed an apparent pK value of 5.0. whereas titration of dehydroretronecine in the presence of excess cysteine resulted in an increase of pK from 5.0 to 8.5. Equilibrium dialysis data indicated appreciable binding of dehydroretronecine to BSA at pH 4.2 and 6°C.
INTRODUCTION
Plants containing the pyrrolizidine alkaloids are distributed worldwide, and include many species in a wide variety of genera including Senecio,Crotalaria, Heliotropium, Trichodesma and Amsinckia (Kingsbury, 1964). Livestock poisoning by consumption of pyrrolizidine alkaloid-containing plants has recently become a major problem in the Pacific northwest and other parts of the United States and the world (Deinzer, Thomson, Burgett & Isaacson, 1977). Consumption by man of foods and herbal medicines contaminated with pyrrolizidine alkaloids has resulted in acute hepatic veno-occlusivediseasewhich progresses to liver cirrhosis (McLean, 1970). In addition, animal experiments have shown that certain pyrrolizidine alkaloids are carcinogenic (Allen, Hsu & Carstens, 1975; Harris & Chen, 1970; Svoboda & Reddy, 1972), mutagenic (Cook & Holt, 1966; Culvenor, Downing, Edgar & Jago, 1969) and teratogenic (Green & Christie, 1961). Dehydroretronecine is the major detectable toxic metabolite of the pyrrolizidine alkaloid, monocrotaline (Hsu, Allen & Chesney, 1973). This pyrrole is responsible for ulceration and atrophy of the gastric mucosa, rhabdomyosarcomas (Allen et al. 1975) and megalohepatocytosis. The discovery of the oncogenic nature of dehydroretronecine prompted investigations of the pyrrole’s interaction with cellular macromolecules in uiuo and in vitro (Hsu, Robertson & Allen, 1976; Hsu, Robertson, Shumaker & Allen, 1975). Their data suggest a direct correlation of the levels of dehydroretronecine binding to cellular macromolecules with the lesions that deveiop in affected organs. Hayashi & Lalich (1968) have shown that injections of either mercaptoethylamine or cysteine will prolong survival and improve the growth rate of rats that receive monocrotaline parenterally. Recently, protective effects of dietary cysteine have been reported in rats affected by pyrrolizidine alkaloid poisoning (Buckmaster, Cheeke & Shull, 1976). This 419
paper presents an account ofthe binding of dehydroretronecine to cysteine and bovine serum albumin (BSA)and the inhibitory effect of dehydroretronecine on a thiol enzyme, yeast alcohol dehydrogenase (ADH). EXPERIMENTAL
Enzyme assay. ADH derived from yeast and nicotinamide adenine dinucleotide (NAD) were purchased from Sigma Chemical Co. (St. Louis, MO.). The activity of yeast ADH was determined by the method of Vallee & Hoch (1955) by measuring the rate of increasein absorbancy at 340 nm, 1 unit being defined as the amount of enzyme that reduced 1 pmol NAD/ min at 25°C under the conditions specified. ADH concentration was determined spectrophotometrically at 280 nm using an extinction coefficient, Ey&%, of 12.6 (Hayes & Valick, 1954) and a molecular weight of 151,000 (Kagi & Vallee, 1960). Inhibition studies. The inhibitory effect of dehydroretronecine on ADH activity was determined by analysis of the initial velocity of the enzyme-mediated reaction in the presence and absence of dehydroretronetine in the reaction mixture. All data were treated by the methods of Lineweaver & Burk (1934) and Hunter & Downs (1945). The ability of cysteine to block the inhibition was determined by adding freshly prepared cysteine solution to the reaction mixture containing dehydroretronecine, substrate and ADH. Spectral
analyses
and
spectrophotbmetric
titrations.
Absorption data were obtained by measuring the appropriate solutions in a Beckman Model DU spectrophotometer with a light path of 1 cm at room temperature. Spectrophotometric titrations were carried out in a Radiometer automatic titrator with a Radiometer Titrigraph type SBR2 and SBUl syringe burette as described by Chu (1971). Samples were withdrawn from the titration vessel at appropriate pH values, and optical density was measured at 24Omn.
P. S. SUN, M.-T.
420
Preparation of dehydroretronecine roretroneciue. Monocrotaline was seed of Crotalaria spectabilis by
and
S.
HSIA,
RESULTS of delrydroretronecine
on enzyme
activity
The inhibitory effect of dehydroretronecine on ADH activity is clearly demonstrated in Fig. 1, which also shows the effect of varying the concentration of dehydroretronecine on ADH activity at a constant concentration of substrate. To examine the type of inhibition produced, the substrate concentration was varied in the presence and absence of dehydroretronecine (7.84 x 10e5 M). The results, plotted according to the method of Lineweaver & Burk (1934) are shown in Fig. 2. The plot indicates that dehydroretronecine has a mixed-type (competitive and noncompetitive) effect on ADH. The inhibition constant (Ki) was found to be 3.38 x lo-’ M under the conditions studied. Blocking
effect
01 cysteine
on enzyme
inhibition
Table 1 shows the results of adding cysteine to the enzyme assay system at zero time in the presence of dehydroretronecine. The inhibitory effect of dehydroTable
1. Efict
of
cysteine
r
0.7 -
0.6 E E $ 0.5 m + 0 0.4 : z I) i 0.3 2 0.2 -
0.1 / 0
L
6 Reoct~on
time.
Fig. 1. ERect of dehydroretronecine on the enzyme activity The reaction was carried out in a pH 7.5 phosphate buffer at 25°C in the absence of dehydroretronecine (0) and in the presence of 7.84 x 10e5 M (0). 2.61 x 10m4~(@) and 3.27 x lo-“ M (m) DR. The conand NAD were centrations of enzyme, ethanol 3.05 x 10m6 M, 2.0 M and 2.5 x IO-” M, respectively. retronecine on ADH activity was blocked completely by the addition of cysteine at a concentration 8-15 times greater than that of dehydroretronecine. Spectrophotometric
titrations
Since dehydroretronecine showed the maximum difference between absorbance in acidic and basic conditions at 240 nm, spectrophotometric titrations were carried out at this wavelength. The titration of dehydroretronecine alone showed an apparent pK of 5.0 (estimated from the half-titration pH) as shown in Fig. 3 (curve d). In the presence of cysteine the pK value shifted from 5.0 to 8.5 (Fig. 3, curve b). Binding between dehydroretronecine and cysteine was evident from the drastic shift in position of the titration curve. of ADH
activity by
Relative enzyme activity (%)
+ cysteinet
100 100 75 100 62
+ cysteinet
100
*The assay conditions were the same as those described in Fig. 2. tFreshly prepared solution of cysteine was added at a final concentration of 3.3 x 10+&4.
fThe dehydroretronecine BThe dehydroretronecine
mln
of ADH.
on the inhibition dehydroretronecine
Incubation mixture* Control Cysteinet Dehydroretronecinej Dehydroretronecinel Dehydroretronecinfi Dehydroretronecine(i
and J. R. ALLEN 0.8
[3H]dehyd-
extracted from the the procedure of Koekemoer & Warren (1950). Dehydroretronecine was prepared from the parent alkaloid, monocrotaline, according to the method of Culvenor, Edgar, Smith & Tweeddale (1970). Dehydroretronecine tritirited at C-7 and C-9 (specific activity 20 &i/mg) was synthesized by a new procedure (M.-T. S. Hsia, unpublished data 1977) based on the reduction of I-formyl-7/?hydroxy-6,7-dihydro-SH-pyrrolizine with NaB3H, (New England Nuclear, Boston, Mass; > 100 mCi/ nmol). Equilibrium dialysis. Equilibrium dialysis was carried out at 6°C according to the method of Rosenberg & Klotz (1960). Detailed procedures were essentially those of Chu (1974) except that tritiated dehydroretronecine was used to calculate the free dehydroretronecine concentration. Radioactivity data were obtained by counting in 10ml of a solution of InstaGel universal liquid scintillation cocktail (Packard Instrument Co., Downers Grove, IL) using a Beckman Model 335 liquid scintillation spectrometer. A molecular weight of 65,000 (Spahr & Edsall, 1964) was used for the calculation of the molar concentration of BSA. Effect
F. S. CHU
concentration was 2.18 x lo-’ concentration was 4.36 x lob5
M. M.
ADH inhibition
by dehydroretronecine
I I/S.
,L
M
Fig. 2. Lineweaver-Burk plots for ADH; control reaction (01: reaction inhibited by 2.61 x 10mJ M dehydroretronecine (0). The reaction was carried out in a pH 7.5 phosphate buffer at 25°C. The concentrations of enzyme. ethanol and NAD were 1.52 x 10W6hl and 2.5 x lo-“M. respectively.
0.5
421
I 1.0 BSA
I
I
I
1.5 2.0 2.5 concentration.
I
I
3.0 3.5 10-4M
I 4.0
Fig. 4. Binding of [‘H]dehydroretronecine with BSA. The data were obtained by equilibrium dialysis at pH 4.2 and 6°C. The C3H]dehydroretronecine concentration was 1.13 x lo-*M(: the BSA concentration varied from 7.6 x 1O-6 to 3.85 x IO-‘M. there was no binding of BSA with dehydroretronecine at pH 7.0.
The binding curve of dehydroretronecine with BSA obtained from equilibrium dialysis in pH 4.2 citratephosphate buffer is shown in Fig. 4. The extrapolated value obtained from the curve indicates that 0.25 mol dehydroretronecine is bound by 1 mol BSA. However, 0.7 l--
Cd)
+ o 0.4-
2
4
6
8
IO
12
PH
Fig. 3. Spectrophotometric titration of cysteine and the dehydroretronecine-cysteine complex: (a) titration data of cysteine alone; (b) titration data of dehydroretronecinecysteine complex; (c) the difference between the data of curves b and a (b - a); (d) titration of free dehydroretronecine. The starting concentration of dehydroretronecine was 3.27 x 10Y4~.
DISCUSSION The inhibition of ADH activity by dehydroretronecine and the binding ofdehydroretronecine with cysteine and BSA have been demonstrated by spectrophotometric analyses and equilibrium dialysis. Inhibition of ADH by p-chloromercuribenzoate has been reported by Snodgrass, Vallee & Hoch (1960), and the inhibition was reversible on addition of excess glutathione. It is possible that the inhibitory effect of dehydroretronecine on ADH was due to the reaction of dehydroretronecine with the thiol groups in the active centre of ADH. The importance of the interaction with thiol compounds to the in ciao toxic action of pyrrolizidine alkaloids can be seen from the protective effect of ip-injected mercaptoethylamine and cysteine against monocrotaline intoxication in rats (Hayashi & Lalich, 1968). This is substantiated by the recent report that dietary cysteine has a protective effect on rats affected by pyrrolizidine alkaloid poisoning (Buckmaster, Cheeke 8c Shull, 1976). However, the protective mechanism of thiol compounds against pyrrolizidine alkaloid intoxication is obscure. Cysteine administration reduced the incidence of chromosomal breakage in Allium roots treated with monocrotaline or allied pyrrolizidine alkaloids (Avanzi, 1961). By analogy with data from radiobiology, it was suggested that cysteine protection against pyrrolizidine alkaloids was operating by lowering the intracellular oxygen tension. As far as we are aware, the present paper contains the first report on the inhibitory effect of a reactive pyrrolizidine alkaloid metabolite, dehydroretronecine, on the activity of an enzyme (ADH) under in vitro conditions, and the reverse effect of cysteine on such enzyme activity. Current literature shows that the alkylation of various cell components is one probable mechanism by which dehydroretronecine produces pathological alterations, whether they be gastric ulceration, hepatocellular necrosis or rhabdomyosar-
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comas. On the basis of our present results, it might be postulated that thiol compounds exert their protective effect by competing with the nucleophilic components of the cells as potential target(s) for alkylation by the pyrrole metabolite(s). The interaction of dehydroretronecine with,. BSA was first studied by spectrophotometric analysrs (P. Sun and F. S. Chu, unpublished data 1976). Positive absorptivity was observed at 22&300nm when dehydroretronecine was incubated with BSA at pH 2.90, 5.22 or 7.20. However, calf-thymus DNA showed negative absorptivity under the same conditions. These results correlated with previous observations (Culvenor et al. 1969; Hsu et al. 1975) that there was a preferential binding of dehydroretronecine to proteins over other cellular components including nucleic acids. Results from equilibrium dialysis indicated no binding to BSA at pH 7.0. However, dehydroretronecine readily bound to BSA at pH 42. The most likely reason for this increased binding at a lower pH is increased protonation of the allylic hydroxyl group(s) in dehydroretronecine with subsequent dehydration, forming a relatively stable allylic carbonium ion which can undergo electrophilic alkylation of macromolecules. Although the significance of the interaction of dehydroretronecine with BSA at neutral pH is not known at present, BSA may serve as a transport agent for this pyrrole, thus providing a constant liberation of the toxic metabolite from the pyrrole-albumin complex to target tissue(s). Acknowledgements-This investigation, published as Primate Center Publication No. 17421, was supported in part by U.S. Public Health Service grants CA-13288, HL-10941 and RR-00167 from the National Institutes of Health.
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