British Jourraal of Haenrafology, 1976, 32, 99.
Induction of Haemolytic Anaemia by Substituted Pheny lhy drazines H. A. ITANO,K. HOSOKAWA AND K. HIROTA Departitieiit of Pathology, Utiivcrsity of California, Sari Dicgo, La Jolla, Calijirtzia
(Received 28 May 1975 ; acceptcdfor pdlicatiori 16]11I1c 1975) SUMMARY. The induction of aiiaciiiia and reticulocytosic by arylliydraziiies was influenced by substitucnts on the beiizeiic ring of plieiiylliydrazine. Arylhydraziiies with ovtho substitucnts, wliicli would hinder tlie binding by liaemoglobiii of a ligaiid derived from the arylliydrazine, resulted in the least aiiaciiiia and reticulocytosis. These results are consistent with a previous finding that liydraziiiobenzoic acid, wliicli did not produce a ferriliaeiiiocliroiiie from ferriliaciiioglobiii, did not induce anaemia. The parallelism between the formation of a fcrrihaciiioglobiii compound aiid the induction of anaemia supports the hypothesis that destabilization of hacmoglobin by the biiidiiig of a ligaiid derived froin tlie aryl portion of an arylliydraziiie is an essential step in arylliydrazinc-induced haeniolytic anaemia. Phciiylliydraziiie is an especially effective chemical inducer of liaeiiiolysis and lieiice is a frequently used reagent for the production of anaemia aiid reticulocytes in laboratory aiiimals. It is well cstablislied tliat plieiiylliydrazine results in prccipitatcs of altcrcd hacnioglobin known as Heiiiz bodies in erythrocytes and that the life span of erythrocytes in the circulation is shorteiicd by the presence of these prccipitates. However, tlic specific mcchanism by which plieiiylliydraziiie causes the formation of Heinz bodies is not fully undcrstood. The expressioiis ‘oxidative denaturation’ and ‘oxidative haemolysis’ have been applied to the chemical and cellular damage induced in haemoglobin and in erythrocytes by phenylhydraziiic and certain other chemicals. Among tlic reactions postulated to have important roles in the oxidative denaturation of liaemoglobin by plieiiylliydrazine are oxidation of thiols (Allen & Jandl, 1961),oxidation of porpliyrin (Lembcrg & Legge, 1942;Kiese & Seipclt, 1943),formation of ferriliaemoglobin (Harley & Mauer, 1960)and formation of an endogenously ligandcd globiii ferrihacniochroiiie (Racliniilewitz ct al, 1973). Itano (1970) suggested that the biiidiiig of a large exogenous ligaiid tliat includes tlie yliciiyl portion of phenylliydrazine might destabilize haemoglobiii and lead to tlie formation of Hcinz bodies. The important role of the aryl component of arylhydraziiies was coiifirnied wlicii it was found that whereas methylplieiiylliydraziiie caused haemolysis in rats, liydrazinobenzoic acid did not (Itano et al, 1974).The hacmolytic activity of these and other riiig-substitutcd yheiiylliydrazines in rabbits is reported here. MATERIALS AND METHODS
2,4-Diinetliylplienylliydraziiie IiydrocliIoride and 2,6-di1iictliylplieiiylliydrazinc liydroCorrespondence: Professor H. A. Itano, fornia 92093, U.S.A.
2402 Boiiner
Hall, University of California, Sail Diego, La Jolla, Cali-
99
I00
H. A. Itano, K. Hosokawa and K. Hirota
chloride were synthesized from the corresponding anilines (Hunsberger et al, 1956), and ethyl 4-hydrazinobenzoate was obtained by the esterification of 4-hydrazinobenzoic acid (Wieland, 1934). Phenylhydrazine hydrochloride was purchased from Eastman Organic Chemicals, Rochester, New York, and all other substituted phenylhydrazines were purchased from Aldrich Chemical Company, Milwaukee, Wisconsin. The compounds were purified by recrystallization from 2 N HC1, except for 2,6-dichlorophenylhydrazine hydrochloride, which was recrystallized from ethanol. The identity of each recrystallized preparation and its purity were confirmed by its nuclear magnetic resonance spectrum, infrared spectrum, melting point, and elemental analyses for carbon, hydrogen, nitrogen and chlorine. Arylliydrazine was prepared for injection by emulsification of I .73 mmol of arylhydrazine hydrochloride in 10 ml of 0.5% aqueous sodium alginate (Sigma Chemical Company, St Louis, Missouri) containing 1.73 mmol of sodium hydroxide. The emulsion was stored at 2°C under highly purified nitrogen. Female domestic rabbits of 2.9k0.6 (SE) kg body weight were used for the induction of anaemia and reticulocytosis. Subcutaneous injections of 52 pmol of test compound per kg of body weight were administered I, 2, 3 and 4 d after a baseline sample of blood was taken. Additional samples of blood were taken 2, 5 and 7 d after the baseline sample. Packed cell volume, reticulocyte count and erythrocyte count were determined. Each packed cell volume was the mean of three determinations,and each reticulocyte count was based on a count of 1000 red blood cells. Control a n i q l s were injected with physiologic saline and with unsubstituted phenylhydrazine dissolved in physiologic saline or emulsified in sodium alginate. RESULTS In all experiments no anaemia or reticulocytosis resulted from physiologic saline, and nearly equal anaemia and reticulocytosis resulted from phenylhydrazine in physiologic saline or in 0.5% aqueous sodium alginate. Parallel results were obtained with determinations of packed cell volume and erythrocyte count; only the former will be reported here. The experimental protocol and results obtained with phenylhydrazine and the isomers of bromophenylhydrazine are shown in Fig I. Among these compounds, phenylhydrazine was the most effective, and 2-bromophenylhydrazinewas the least effective. 3-Bromophenylhydrazine was intermediate in its effect, as was the 4- isomer. As in Fig I, differences among compounds were evident on each of days 2 , 5 and 7 in the other experiments; therefore in reporting the results with' other compounds, only the results on day 5 , when both anaemia and reticulocytosis were maximal in nearly all animals, will be shown. In Fig I the packed cell volume is the per cent of total blood volume. In Figs 2-4 a relative value for each animal, which was calculated as IOO times the ratio of the packed cell volume on day 5 to that on the baseline day (day o), is shown. Relative packed cell volumes and reticulocyte counts obtained with the isomers of chlorophenylhydrazineand dichlorophenylhydrazine in a concurrently injected group of animals are shown in Fig 2 . Unsubstituted phenylhydrazine was the most effective inducer of anaemia and reticulocytosis. The relative effectiveness of the substituted compounds corresponded primarily with position and secondarily with number of substituents. The most effective of the substituted compounds were those with one or two chlorine atoms at ring positions meta (3- or 5-) or para (4-)to the
Induction of Haemolytic Anaemia
I01
0
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i
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1
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7
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4 r Days
FIG I. Packed cell volumes and reticulocyte counts in rabbits injected with emulsions of arylhydrazines in sodium alginate. 0 , Phenylhydrazine; 0,2-bromophenylhydrazine; A , 3-bromophenylhydrazine. For each kg of body weight, 52 pmol of arylhydrazine were injccted on each of four successive days as indicated. Not plotted because of closeness to the results with 3-bromophenylhydrazine were the results with 4-bromophenylhydrazine. Results with the latter on days 0, 2 , 5 and 7, respectively, were as follows: packed cell volume, 40.7, 28.2,21.1and 25.8%; reticulocyte count, 5.4, 16.7, 81.6 and 33.8%.
'1'""
1 I
FIG 2. Relative packed cell volumes (day j/day 0 ) and reticulocyte counts in rabbits after the injection of unsubstituted phenylhydrazine and of chloro-substituted phenylhydrazines according to the protocol of Fig I.
I02
H. A. Itaito, K. Hosokawa
arid
K. Hirota
liydrazino group, namely tlie 3- or 4-chlor0- and the 3,4- or 3 ,s-dichloroplicnylliydraziiies. Compounds with oiie ortho (z-) chlorine atom were less effective, and of these compounds, those with a second chlorine atom either meta or para to tlie liydraziiio group werc less effective than monosubstituted 2-chlorophenylliydrazine. 2,6-Dichloropheiiylliydrazine, which has two ortho cliloriiie atoms, caused neither anaemia nor reticulocytosis. Compounds with methyl and carboxy substituents, some of wliich wcrc previously tested in rats (Itano et a!, 1974, were compared in the experiment summarized by Fig 3. Methylpheiiylhydrazine (z- or 4-) was as effective as uiisubstituted phenylhydraziiie, but 2,4djiiiethylpliciiylliydraziiie was less effcctive. 2,6-DiniethyIphe1iylhydrazine, wliich has two ortho methyl substituents, was ineffective. A carboxy substituent either ortho or para to the liydraziiio group abolished tlie haemolytic effect of phenylhydrazine in 2- or 4-hydrazinobeiizoic acid, respectively. On the other hand, an ester of 4-liydrazinobeiizoic acid resulted in mild anaemia and reticulocytosis.
- loo -80
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C
-60 0 c
G
-40 $ ._ c
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-20 0
n
n
n
FIG3 . Relativc packed cell volumes (day S/day 0)and reticulocyte counts ofrabbits after the injection of unsubstituted phcnylhydrazine and of methyl-, dimethyl-, carboxy- and carboxyethyl-substituted phenylhydrazincs according to thc protocol of Fig I.
Compounds with one or two ortho substituciits were compared in a triplicate series of animals in the experiment of Fig 4.This experiment shows the degree of reproducibility of the relativc packed cell volume on day 5 within sets of three animals treated in identical manner. The ineffectiveness of 2,6-dichloro- and 2,6-dimetliylphenylliydrazine was confirmed. 2,3 ,~,6-Tetrafluoropheiiylhydrazine,which has fluorine atoms at ring positions orbho (2- and 6-) to the liydraziiio group, resulted in a mild anaemia. DISCUSSION Phciiylhydrazinc is oxidized by ferricyanide or oxygen to plienyldiazene (C,H,N=NH), which reacts with ferrihaemoglobin to produce a compound with the optical spectrum of a ferrihaemochrome (Itano & Robinson, 1961 ; Itano, 1970). Each of the three riiig-substituted isomers of methylplieiiylhydrazine is also oxidized to the corresponding diazeiie (Maniien &
Itididon
of Hnernolytic Aiiaenzia
103
Itano, 1973), and cach of tlie latter reacts with ferrihaemoglobiii to produce a ferrihaemochrome-likc coinpouiid (Itaiio ct al, 1974). The ovtho and yavn isomers of hydraziiiobeiizoic acid are also oxidized to tlicir diazciiyl products, but tlie latter do iiot form ferriliaeiiiochromes. The induction of anaemia by an arylhydraziiie paralleled tlie formation of ferriliaemochromes by its diazciic in that whercas cach mcthylpheiiylhydrazinc iiiduced haemolytic anaemia, 4-hydraziiiobenzoic acid did not (Itaiio ct nl, 1974). These rcsults with methylplienylhydraziiie and hydrazinobcnzoic acid in rats, which were coiifiriiicd in rabbits by the prcsciit study, showed an cffcct ofcharge, but not of ring position, of substitueiits. The prcseiit study established a position effect in that substitueiits ovtho to the liydraziiio group exerted tlie greatest iiiliibitory effcct on tlie haemolytic action of arylhydrazines. Substituciits in this position would result in tlie greatest steric hiiidraiicc to the formation of a bond betwecii an
FIG 4. Rclativc packcd ccll voluiiics (day day 0 ) of rabbits aftcr thc iiijcctioii of unsubstituted phenylhydraziiic aiid of 2- and z,6-substituted phenylhydrazincs according to the protocol of Fig I. Thrce animals wcrc injected with cach coinpouiid cxccpt unsubstitutcd phcnylhydrazine.
aryldiazeiie and tlie iron atom of the ferrihaem group (Itaiio, 1970). Two ovtho chlorine atoms or iiictliyl groups prcvciited tlie induction of anaemia, but the smaller fluorine atom in the same ring positioiis did iiot. The fiiidiiig that an ester of 4-liydrazinobeiizoic acid iiiduced a mild anaemia and reticulocytosis iiidicatcs that the iiieffcctiveiiess of 4-liydraziiiobeiizoic acid resulted from tlie negative charge aiid not the size or tlic position on the beiizciic ring of its carboxy group. Our fiiidiiigs are coiisisteiit with tlic hypothesis (Itaiio, 1970) that the binding of pheiiyldiazciie by ferriliaemoglobin plays an important role in the induction of haemolytic anaemia by pheiiylliydraziiie. Just as an inherited amino acid substitution in tlie haem pockct destabilizes haemoglobiii in coiigciiital Heiiiz body anaemia (Pcrutz & Lchmaiiii, 1968), the large exogenous aryl ligaiid derivcd froin an arylhydraziiie is postulated to result in conformatioiial instability by breaking lion-polar contacts betwceii haem aiid its globiii pockct.
104
H. A. Itano, K. Hosokawa and K. Hivota
The formation of ferrihaemoglobin is an essential preliminary event to the formation of a ferrihaemochrome;however, the former reaction does not depend on the specific structure of an arylhydrazine and does not destabilize the molecule appreciably. W e suggest that the instability induced by the exogenous ligand of aryldiazene-ferrihaemochrome exposes susceptible groups such as the thiols of globin and the porphyrin of haem to oxidation. Thus, although the latter reactions are important events in phenylhydrazine-induced oxidative denaturation of haemoglobin, these events are considered to be secondary to the structural destabilization of ferrihaemoglobin by the presence in the haem pocket of a large ligand derived from phenylhydrazine. The primary role of destabilization in the formation of Heinz bodies is supported by the fact that congenital Heinz body anaemia is usually observed in the absence of drugs with oxidant action (Carrel1& Lehman, 1969). ACKNOWLEDGMENT
This investigation was supported in part by Grants GM 17702 and AM 14982 from the National Institutes of Health. REFERENCES ALLEN, D.W. & JANDL, J.H. (1961) Oxidative hemolysis and precipitation of hemoglobin. 11. Role of thiols in oxidant drug action. Journal of Clinical Investigation, 40, 454. CARRELL, R.W. & LEHMANN,H. (1969) The unstable haemoglobin haemolytic anaemias. Seminars in Hematology, 6, 116. HARLEY, J.D. & MAUER, A.M. (1960) Studies on the formation of Heinz bodies. I. Methemoglobin production and oxyhemoglobin destruction. Blood, 16, 1722. HUNSBERGER, M., SHAW,E.R., FUGGER, J., KETCHAM, R. & LEDNICER,D. (1956) The preparation of substituted hydrazines. IV. Arylhydrazines via conventional methods. Journal of Organic Chemistry, 21, 394.
ITANO, H.A. (1970) Phenyldiimide, hemoglobin, and Heinz bodies. Proceedings of the National Academy of Sciences ofthe United States of America, 67,485. ITANO,H.A., HOLLISTER, D.W., FOGARTY, W. M., JR & MANNEN, S. (1974) Effect of ring substitution on the hemolytic action of arylhydrazines. Proceedings of the Society for Experimental Biology and Medicine, 147, 656.
ITANO,H.A. & ROBINSON, E.A. (1961) Evidence for coordination of monophenyl diimide with heme proteins. Journal of the American Chemical Society, 83, 3339. KIESE, J. & SEIPELT, L. (1943) BiIdung und Elimination von Verdoglobinen. Naunyu-Schmiedebergs Archiv fiir Experimentelk Pathologie und Pharmakologie, zoo, 648.
LEMBERG, R. & LEGGE, J.W. (1942) Intracorpuscular bile pigment formation. Australian Journal of Experimental Biology and Medical Science, 20, 65. MANNEN, S. & ITANO,H.A. (1973) Stoichiometry of the oxidation of arylhydrazines with ferricyanide. Tetrahedron, 29, 3497. PERUTZ,M.F. & LEHMANN,H. (1968) Molecular pathology ofhuman haemoglobin. Nature, 219,902. RACHMILEWITZ, E.A., PEISACH, J. & BLUMBERG, W.E. Personal communication, cited in NAGEL, R.L. & RANNEY,H.M. (1973) Drug-induced oxidative denaturation of hemoglobin. Seminars in Hematology, 10, 269. WIELAND,H. (1934) Uber das Auftreten freier Radikale bei chemischen Reactionen. VIII. Justus Liebigs Annalen der Chemie, 514, 145.
Induction of Haemolytic Anaemia by Substituted Pheny lhy drazines H. A. ITANO,K. HOSOKAWA AND K. HIROTA Departitieiit of Pathology, Utiivcrsity of California, Sari Dicgo, La Jolla, Calijirtzia
(Received 28 May 1975 ; acceptcdfor pdlicatiori 16]11I1c 1975) SUMMARY. The induction of aiiaciiiia and reticulocytosic by arylliydraziiies was influenced by substitucnts on the beiizeiic ring of plieiiylliydrazine. Arylhydraziiies with ovtho substitucnts, wliicli would hinder tlie binding by liaemoglobiii of a ligaiid derived from the arylliydrazine, resulted in the least aiiaciiiia and reticulocytosis. These results are consistent with a previous finding that liydraziiiobenzoic acid, wliicli did not produce a ferriliaeiiiocliroiiie from ferriliaciiioglobiii, did not induce anaemia. The parallelism between the formation of a fcrrihaciiioglobiii compound aiid the induction of anaemia supports the hypothesis that destabilization of hacmoglobin by the biiidiiig of a ligaiid derived froin tlie aryl portion of an arylliydraziiie is an essential step in arylliydrazinc-induced haeniolytic anaemia. Phciiylliydraziiie is an especially effective chemical inducer of liaeiiiolysis and lieiice is a frequently used reagent for the production of anaemia aiid reticulocytes in laboratory aiiimals. It is well cstablislied tliat plieiiylliydrazine results in prccipitatcs of altcrcd hacnioglobin known as Heiiiz bodies in erythrocytes and that the life span of erythrocytes in the circulation is shorteiicd by the presence of these prccipitates. However, tlic specific mcchanism by which plieiiylliydraziiie causes the formation of Heinz bodies is not fully undcrstood. The expressioiis ‘oxidative denaturation’ and ‘oxidative haemolysis’ have been applied to the chemical and cellular damage induced in haemoglobin and in erythrocytes by phenylhydraziiic and certain other chemicals. Among tlic reactions postulated to have important roles in the oxidative denaturation of liaemoglobin by plieiiylliydrazine are oxidation of thiols (Allen & Jandl, 1961),oxidation of porpliyrin (Lembcrg & Legge, 1942;Kiese & Seipclt, 1943),formation of ferriliaemoglobin (Harley & Mauer, 1960)and formation of an endogenously ligandcd globiii ferrihacniochroiiie (Racliniilewitz ct al, 1973). Itano (1970) suggested that the biiidiiig of a large exogenous ligaiid tliat includes tlie yliciiyl portion of phenylliydrazine might destabilize haemoglobiii and lead to tlie formation of Hcinz bodies. The important role of the aryl component of arylhydraziiies was coiifirnied wlicii it was found that whereas methylplieiiylliydraziiie caused haemolysis in rats, liydrazinobenzoic acid did not (Itano et al, 1974).The hacmolytic activity of these and other riiig-substitutcd yheiiylliydrazines in rabbits is reported here. MATERIALS AND METHODS
2,4-Diinetliylplienylliydraziiie IiydrocliIoride and 2,6-di1iictliylplieiiylliydrazinc liydroCorrespondence: Professor H. A. Itano, fornia 92093, U.S.A.
2402 Boiiner
Hall, University of California, Sail Diego, La Jolla, Cali-
99
I00
H. A. Itano, K. Hosokawa and K. Hirota
chloride were synthesized from the corresponding anilines (Hunsberger et al, 1956), and ethyl 4-hydrazinobenzoate was obtained by the esterification of 4-hydrazinobenzoic acid (Wieland, 1934). Phenylhydrazine hydrochloride was purchased from Eastman Organic Chemicals, Rochester, New York, and all other substituted phenylhydrazines were purchased from Aldrich Chemical Company, Milwaukee, Wisconsin. The compounds were purified by recrystallization from 2 N HC1, except for 2,6-dichlorophenylhydrazine hydrochloride, which was recrystallized from ethanol. The identity of each recrystallized preparation and its purity were confirmed by its nuclear magnetic resonance spectrum, infrared spectrum, melting point, and elemental analyses for carbon, hydrogen, nitrogen and chlorine. Arylliydrazine was prepared for injection by emulsification of I .73 mmol of arylhydrazine hydrochloride in 10 ml of 0.5% aqueous sodium alginate (Sigma Chemical Company, St Louis, Missouri) containing 1.73 mmol of sodium hydroxide. The emulsion was stored at 2°C under highly purified nitrogen. Female domestic rabbits of 2.9k0.6 (SE) kg body weight were used for the induction of anaemia and reticulocytosis. Subcutaneous injections of 52 pmol of test compound per kg of body weight were administered I, 2, 3 and 4 d after a baseline sample of blood was taken. Additional samples of blood were taken 2, 5 and 7 d after the baseline sample. Packed cell volume, reticulocyte count and erythrocyte count were determined. Each packed cell volume was the mean of three determinations,and each reticulocyte count was based on a count of 1000 red blood cells. Control a n i q l s were injected with physiologic saline and with unsubstituted phenylhydrazine dissolved in physiologic saline or emulsified in sodium alginate. RESULTS In all experiments no anaemia or reticulocytosis resulted from physiologic saline, and nearly equal anaemia and reticulocytosis resulted from phenylhydrazine in physiologic saline or in 0.5% aqueous sodium alginate. Parallel results were obtained with determinations of packed cell volume and erythrocyte count; only the former will be reported here. The experimental protocol and results obtained with phenylhydrazine and the isomers of bromophenylhydrazine are shown in Fig I. Among these compounds, phenylhydrazine was the most effective, and 2-bromophenylhydrazinewas the least effective. 3-Bromophenylhydrazine was intermediate in its effect, as was the 4- isomer. As in Fig I, differences among compounds were evident on each of days 2 , 5 and 7 in the other experiments; therefore in reporting the results with' other compounds, only the results on day 5 , when both anaemia and reticulocytosis were maximal in nearly all animals, will be shown. In Fig I the packed cell volume is the per cent of total blood volume. In Figs 2-4 a relative value for each animal, which was calculated as IOO times the ratio of the packed cell volume on day 5 to that on the baseline day (day o), is shown. Relative packed cell volumes and reticulocyte counts obtained with the isomers of chlorophenylhydrazineand dichlorophenylhydrazine in a concurrently injected group of animals are shown in Fig 2 . Unsubstituted phenylhydrazine was the most effective inducer of anaemia and reticulocytosis. The relative effectiveness of the substituted compounds corresponded primarily with position and secondarily with number of substituents. The most effective of the substituted compounds were those with one or two chlorine atoms at ring positions meta (3- or 5-) or para (4-)to the
Induction of Haemolytic Anaemia
I01
0
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1
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i
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i
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1
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4 r Days
FIG I. Packed cell volumes and reticulocyte counts in rabbits injected with emulsions of arylhydrazines in sodium alginate. 0 , Phenylhydrazine; 0,2-bromophenylhydrazine; A , 3-bromophenylhydrazine. For each kg of body weight, 52 pmol of arylhydrazine were injccted on each of four successive days as indicated. Not plotted because of closeness to the results with 3-bromophenylhydrazine were the results with 4-bromophenylhydrazine. Results with the latter on days 0, 2 , 5 and 7, respectively, were as follows: packed cell volume, 40.7, 28.2,21.1and 25.8%; reticulocyte count, 5.4, 16.7, 81.6 and 33.8%.
'1'""
1 I
FIG 2. Relative packed cell volumes (day j/day 0 ) and reticulocyte counts in rabbits after the injection of unsubstituted phenylhydrazine and of chloro-substituted phenylhydrazines according to the protocol of Fig I.
I02
H. A. Itaito, K. Hosokawa
arid
K. Hirota
liydrazino group, namely tlie 3- or 4-chlor0- and the 3,4- or 3 ,s-dichloroplicnylliydraziiies. Compounds with oiie ortho (z-) chlorine atom were less effective, and of these compounds, those with a second chlorine atom either meta or para to tlie liydraziiio group werc less effective than monosubstituted 2-chlorophenylliydrazine. 2,6-Dichloropheiiylliydrazine, which has two ortho cliloriiie atoms, caused neither anaemia nor reticulocytosis. Compounds with methyl and carboxy substituents, some of wliich wcrc previously tested in rats (Itano et a!, 1974, were compared in the experiment summarized by Fig 3. Methylpheiiylhydrazine (z- or 4-) was as effective as uiisubstituted phenylhydraziiie, but 2,4djiiiethylpliciiylliydraziiie was less effcctive. 2,6-DiniethyIphe1iylhydrazine, wliich has two ortho methyl substituents, was ineffective. A carboxy substituent either ortho or para to the liydraziiio group abolished tlie haemolytic effect of phenylhydrazine in 2- or 4-hydrazinobeiizoic acid, respectively. On the other hand, an ester of 4-liydrazinobeiizoic acid resulted in mild anaemia and reticulocytosis.
- loo -80
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C
-60 0 c
G
-40 $ ._ c
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-20 0
n
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FIG3 . Relativc packed cell volumes (day S/day 0)and reticulocyte counts ofrabbits after the injection of unsubstituted phcnylhydrazine and of methyl-, dimethyl-, carboxy- and carboxyethyl-substituted phenylhydrazincs according to thc protocol of Fig I.
Compounds with one or two ortho substituciits were compared in a triplicate series of animals in the experiment of Fig 4.This experiment shows the degree of reproducibility of the relativc packed cell volume on day 5 within sets of three animals treated in identical manner. The ineffectiveness of 2,6-dichloro- and 2,6-dimetliylphenylliydrazine was confirmed. 2,3 ,~,6-Tetrafluoropheiiylhydrazine,which has fluorine atoms at ring positions orbho (2- and 6-) to the liydraziiio group, resulted in a mild anaemia. DISCUSSION Phciiylhydrazinc is oxidized by ferricyanide or oxygen to plienyldiazene (C,H,N=NH), which reacts with ferrihaemoglobin to produce a compound with the optical spectrum of a ferrihaemochrome (Itano & Robinson, 1961 ; Itano, 1970). Each of the three riiig-substituted isomers of methylplieiiylhydrazine is also oxidized to the corresponding diazeiie (Maniien &
Itididon
of Hnernolytic Aiiaenzia
103
Itano, 1973), and cach of tlie latter reacts with ferrihaemoglobiii to produce a ferrihaemochrome-likc coinpouiid (Itaiio ct al, 1974). The ovtho and yavn isomers of hydraziiiobeiizoic acid are also oxidized to tlicir diazciiyl products, but tlie latter do iiot form ferriliaeiiiochromes. The induction of anaemia by an arylhydraziiie paralleled tlie formation of ferriliaemochromes by its diazciic in that whercas cach mcthylpheiiylhydrazinc iiiduced haemolytic anaemia, 4-hydraziiiobenzoic acid did not (Itaiio ct nl, 1974). These rcsults with methylplienylhydraziiie and hydrazinobcnzoic acid in rats, which were coiifiriiicd in rabbits by the prcsciit study, showed an cffcct ofcharge, but not of ring position, of substitueiits. The prcseiit study established a position effect in that substitueiits ovtho to the liydraziiio group exerted tlie greatest iiiliibitory effcct on tlie haemolytic action of arylhydrazines. Substituciits in this position would result in tlie greatest steric hiiidraiicc to the formation of a bond betwecii an
FIG 4. Rclativc packcd ccll voluiiics (day day 0 ) of rabbits aftcr thc iiijcctioii of unsubstituted phenylhydraziiic aiid of 2- and z,6-substituted phenylhydrazincs according to the protocol of Fig I. Thrce animals wcrc injected with cach coinpouiid cxccpt unsubstitutcd phcnylhydrazine.
aryldiazeiie and tlie iron atom of the ferrihaem group (Itaiio, 1970). Two ovtho chlorine atoms or iiictliyl groups prcvciited tlie induction of anaemia, but the smaller fluorine atom in the same ring positioiis did iiot. The fiiidiiig that an ester of 4-liydrazinobeiizoic acid iiiduced a mild anaemia and reticulocytosis iiidicatcs that the iiieffcctiveiiess of 4-liydraziiiobeiizoic acid resulted from tlie negative charge aiid not the size or tlic position on the beiizciic ring of its carboxy group. Our fiiidiiigs are coiisisteiit with tlic hypothesis (Itaiio, 1970) that the binding of pheiiyldiazciie by ferriliaemoglobin plays an important role in the induction of haemolytic anaemia by pheiiylliydraziiie. Just as an inherited amino acid substitution in tlie haem pockct destabilizes haemoglobiii in coiigciiital Heiiiz body anaemia (Pcrutz & Lchmaiiii, 1968), the large exogenous aryl ligaiid derivcd froin an arylhydraziiie is postulated to result in conformatioiial instability by breaking lion-polar contacts betwceii haem aiid its globiii pockct.
104
H. A. Itano, K. Hosokawa and K. Hivota
The formation of ferrihaemoglobin is an essential preliminary event to the formation of a ferrihaemochrome;however, the former reaction does not depend on the specific structure of an arylhydrazine and does not destabilize the molecule appreciably. W e suggest that the instability induced by the exogenous ligand of aryldiazene-ferrihaemochrome exposes susceptible groups such as the thiols of globin and the porphyrin of haem to oxidation. Thus, although the latter reactions are important events in phenylhydrazine-induced oxidative denaturation of haemoglobin, these events are considered to be secondary to the structural destabilization of ferrihaemoglobin by the presence in the haem pocket of a large ligand derived from phenylhydrazine. The primary role of destabilization in the formation of Heinz bodies is supported by the fact that congenital Heinz body anaemia is usually observed in the absence of drugs with oxidant action (Carrel1& Lehman, 1969). ACKNOWLEDGMENT
This investigation was supported in part by Grants GM 17702 and AM 14982 from the National Institutes of Health. REFERENCES ALLEN, D.W. & JANDL, J.H. (1961) Oxidative hemolysis and precipitation of hemoglobin. 11. Role of thiols in oxidant drug action. Journal of Clinical Investigation, 40, 454. CARRELL, R.W. & LEHMANN,H. (1969) The unstable haemoglobin haemolytic anaemias. Seminars in Hematology, 6, 116. HARLEY, J.D. & MAUER, A.M. (1960) Studies on the formation of Heinz bodies. I. Methemoglobin production and oxyhemoglobin destruction. Blood, 16, 1722. HUNSBERGER, M., SHAW,E.R., FUGGER, J., KETCHAM, R. & LEDNICER,D. (1956) The preparation of substituted hydrazines. IV. Arylhydrazines via conventional methods. Journal of Organic Chemistry, 21, 394.
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