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Hemolytic effect of phenylhydrazine during amphibian metamorphosis
DEVELOPMENTAL
BIOLOGY
Hemolytic
27, 406418
(1972)
Effect of Phenylhydrazine Metamorphosis GUSTAVO
Department
of Chemistry,
during
Amphibian
FLORES ’ AND EARL FRIEDEN* Florida Accepted
State
Uniuersity,
November
Tallahassee,
Florida
32306
9, 1971
The correlation between the spectral changes in hemoglobin and the severity of anemia induced by phenylhydrazine treatment was studied for the differential sensitivity of amphibians to the drug. Froglets were the most sensitive to phenylhydrazine, follrwed by prometamorphic tadpoles, adult frogs, metamorphic climax tadpoles, and triiodothyronine-treated tadpoles. The different sensitivities to the hemolytic action of the drug in these animals was rationalized in terms of accessibility, uptake, and detoxication of phenylhydrazine, and a different rate of removal of damaged cells. Postmetamorphic responses were noted for the low uptake of phenylhydrazine by erythrocytes and the loss of facilitated diffusion of 3-0-methylglucose by the erythrocytes of the adult frog. The rate of regeneration of erythrocytes (erythropoiesis) after phenylhydrazine treatment followed an order almost opposite to that observed in the hemolytic process. Thus, tadpoles at metamorphic climax showed the maximum rate of regeneration, followed by T,-treated tadpoles, adult frogs, froglets, and prometamorphic tadpoles. Erythropoiesis immediately following the hemolytic phase did not reveal any abnormal hemoglobins. Survival after exposure to phenylhydrazine followed the same order described for hemolytic susceptibility, i.e., froglets had the highest percent survival. Tadpoles immersed in 2,4-dinitrophenol were more sensitive to the lethal effects of phenylhydrazine.
box turtle with more than 90% of its Hb Vertebrate species completely lacking present as methemoglobin has also been erythrocytes and hemoglobin (Hb) have reported (Sullivan and Riggs, 1964). Cerbeen reported (Rudd, 1954). The main tain amphibians and other species are respiratory compensation in a family of characterized by seasonal changes in Hb fishes (Chaemichthyidae) appears to be concentration which may be adaptive (Anan increase of blood volume and high cu- drew, 1965; Theil, 1967; Friedman et aZ., taneous respiration (Hemmingsen and 1969; Banerjee and Banerjee, 1969). We have reported the unusual sensitivDouglas, 1970). Occasionally, amphibians ity of amphibian red blood cells to phenylwithout erythrocytes have been found hydrazine and other hydrazine derivatives (DeGraaf, 1957; Ewer, 1959). Nate (per(Flores and Frieden, 1968, 1970). A low sonal communication) has found mutant dose of phenylhydrazine eliminates the tadpoles which are completely lacking in erythrocytes and Hb of Ranu tadpoles in Hb. This has been confirmed in our laboraa short time, and the animals survive withtory with observations that some tadpoles out obvious difficulty. A direct correlation and adult bullfrogs survived with blood Hb levels as low as 1.0 gm/lOO ml (Flores, between the spectral changes of the Hb 1969). The survival of a specimen of the molecule observed in vitro and the in vivo effect of hemolytically active hydrazines 1 Present address: Oregon State University, Enwas also demonstrated (Flores and Frievironmental Health Science Center, Corvallis, den, 1970). The experimental production Oregon 97331. of anemic newts (Grass0 and Shephard, * Reprint requests should be sent to: Earl Frieden, 1968), anemic frogs (Meints, 1969) and Department of Chemistry, Florida State University, Tallahassee, Florida 32306. anemic fish (Cameron and Wohlschlag, INTRODUCTION
Copyright
0 1972 by Academic
Press, Inc.
406
FLORES
AND FRIEDEN
Hemolysis
1969) have also been reported. The numerous biochemical changes that take place during amphibian metamorphosis and their adaptive implications have been reviewed most recently by Frieden and Just (1970). Prominent among those changes is the shift in the main erythropoietic organ of the larval tadpole as compared to the adult frog (Maniatis and Ingram, 1971a-c). This shift appears to result in the production of a new adult red blood cell line differing from the larval erythrocyte in several characteristics including hemoglobin patterns, osmotic fragility, size and shape of the erythrocytes (Herner and Frieden, 1961; Hollyfield, 1966; Moss and Ingram, 1968a, b; Dewitt, 1968; Benbassat, 1970; Maniatis and Ingram, 1971a-c). Therefore we decided to explore the susceptibility of amphibian erythrocytes to the hemolytic action of phenylhydrazine (1) during spontaneous metamorphosis, (2) in the triiodothyronine-(T,)treated tadpole, and (3) in the adult frog. We also sought to correlate any differential susceptibility to phenylhydrazine with the developmental stage of the animal and to study the regenerative phase (erythropoiesis) taking place immediately after the hemolytic phase in phenylhydrazine-treated amphibians and identify their Hb patterns during recovery. METHODS
AND
MATERIALS
Phenylhydrazine hydrochloride was obtained from the Fischer Scientific Co., St. Louis, Missouri, and was recrystallized from concentrated hydrochloric acid every 3-4 months. The crystals were kept in a desiccator at -15°C to minimize oxidation. L-Triiodothyronine (T,) was donated by Smith, Kline, and French Labs., Philadelphia, Pennsylvania. Cyanmethemoglobin reagent and standard were purchased from Hycel, Inc., Houston, Texas. The chemicals for polyacrylamide gel electrophoresis were obtained from Eastman Organic Chemicals, Roch-
and Anumn
Metamorphosis
407
ester, New York. 3-O-methy114C-n-glucase was purchased from New England Nuclear, Boston, Massachusetts. Rana catesbeiana tadpoles and frogs were purchased from the Lemberger Co., Oshkosh, Wisconsin. Froglets were selected from R. catesbeiana tadpoles which had completed metamorphosis within 1 month of their use. R. grylio and R. pipiens were collected from local ponds. All animals were starved at least 1 day before and during the experiment. The laboratory temperature was held at 22 + 2°C. The metamorphic development of bullfrog tadpoles was classified according to the stage criteria of Taylor and Kollros (1946). Prometamorphosis refers to stages X to XX and metamorphic climax to stages XX to XXV (for further details see review of Frieden and Just, 1970). Blood was obtained by exposing the heart of the tadpole and froglets, removing any tissue fluid and cutting or piercing the arterial trunk and allowing the blood to flow into a heparinized pipette. For adult frogs, the samples were obtained by heart puncture using a 5-ml syringe fitted with a 20-gauge needle. The concentration of Hb was determined by the cyanmethemoglobin method as described earlier (Flores and Frieden, 1970). Individual samples of blood were collected in heparinized (0.01%) amphibian Ringer’s (Rugh, 1962). These samples were diluted as necessary and the erythrocytes counted in a hemacytometer (Neubauer by C. A. Hauser and Sons, Philadelphia). The separation of the hemoglobins by polyacrylamide gel disc electrophoresis using lysates of amphibian erythrocytes was performed as described by Moss and Ingram (1968a). Experimental design. The in vivo treatment consisted of an intraperitoneal injection of 35 mM phenylhydrazine (1 or 2 injections of 5 &gm) or immersion of the animals in 35 PM phenylhydrazine in tap water, 6 animals per liter. All
408
DEVELOPMENTAL
BIOLOGY
phenylhydrazine solutions were brought to pH 7.0-7.4 with dilute NaOH. T, was injected intraperitoneally-0.1 nmoles or 0.5 nmole per gram of tadpole. For the in vitro incubation, individual samples of frog blood or pooled samples (3 to 10) of tadpole or froglet blood were collected in heparinized (0.01% heparin) amphibian Ringer’s solution (Rugh, 1948) and washed 3 times with amphibian Ringers. A 1: 100 suspension of erythrocytes in amphibian Ringer’s was incubated for 5 min at 18 + 1°C with the indicated concentration of phenylhydrazine. The cells were centrifuged at maximum speed in a clinical centrifuge for 1 min and washed with 30 volumes of amphibian Ringers. The cells were lysed in one-half amphibian Ringer’s by freezing and thawing 3 times. Another aliquot of erythrocytes was lysed before incubation with phenylhydrazine. All the samples were adjusted to a final composition of amphibian Ringer’s and centrifuged for 10 min and the clear supernatant was taken for spectral analysis using a Beckman DB spectrophotometer. RESULTS
Hemolytic Effect of Phenylhydrazine during Spontaneous metamorphosis Figure 1 shows the effect of phenylhydrazine administered by immersion or intraperitoneal injection to tadpoles at various stages of metamorphosis. The immersion data, Fig. lA, indicate that the more advanced the metamorphosis the less sensitive were the tadpoles to the hemolytic action of the drug. Thus, 4 days after initiation of treatment all groups except the froglets (stage XXV) showed a minimum Hb concentration, with 98-99% hemolysis in tadpoles between stages X and XIX, over 97% hemolysis in the group at stages XX and XXI, and 90% hemolysis in animals between stages XXII and XXIV. Froglets were relatively insensitive to the immersion treatment as indicated by the
VOLUME
21, 1972
fact that less than 50% hemolysis was produced 2 days after immersion of the experimental group in 35 &f phenylhydrazine. The decreasing hemolytic effect of the drug while metamorphosis proceeds may be due to the decreased availability of phenylhydrazine to the blood and other tissues because of thickening of the skin and loss of the contribution of the gills to the absorption of the drug (Frieden and Just, 1970). The effect of intraperitoneal injection of phenylhydrazine in tadpoles at various stages of spontaneous metamorphosis is shown in Fig. 1B. Two injections of 25 pg (0.175 pmole) of phenylhydrazine per gram body weight were given to the animals, with an interval of 24 hr after the first injection. The maximum hemolytic effect is reached 4 days after the first injection. A fast hemolytic phase (represented by experimental groups 2 and 4 days after administration of the drug) is followed by a slow red cell regenerative phase (represented by the group 7 days after initiation of the experiments). Thus with the exception of froglets for which the highest hemolytic effect of the drug is observed, hemolysis is greater at the lower metamorphic stages, while the regenerative phase is faster with more advanced metamorphosis. Effect of Phenylhydrazine Injection Froglets and Adult Frogs
in
Table 1 shows the effect of intraperitoneal injection of phenylhydrazine into young froglets and in two different adult species. As in Fig. 1, the injection of phenylhydrazine proved to be more effective in producing a fast hemolytic response in froglets than it did in tadpoles. Concentrations of Hb as low as 3% relative to controls were obtained as early as 1 day after injection of phenylhydrazine. In the adult bullfrog (second and third groups in Table l), the hemolytic process
FLORES
f 8 2 w 0
AND FRIEDEN
Hemolysis
and Anumn
409
Metamorphosis
0.8 -
0.6-
7 2 4 DAYS FIG. 1. Effect of immersion and intraperitoneal injection of phenylhydrazine during spontaneous metamorphosis. Each experimental value (vertical bars) represents the average of 7-10 animals. Vertical lines indicate = one standard deviation from average. 0, Stages X-XIII (4.0 + 0.5); N, stages XVIII and XIX (4.5 + 0.4); 0, stages XX and XXI (6.4 i 0.6); I, stages XXII-XXIV (7.2 + 0.8);a, stages XXV (5.5 + 0.5). The grams per 100 ml (gram 9%) Hb for the controls of each experimental group are given in parentheses and represent the average of 7-10 analyses with standard deviation. Left, Fig. 1A: Immersion of tadpoles in 35 FM phenylhydrazine solution. Six animals per liter of solution were immersed for 24 hrs at room temperature (23 + 1°C). Samples were collected 2, 4, and 7 days after initiation of experiment. Right, Fig. 1B: Intraperitoneal injection of phenylhydrazine. The tadpoles were injected twice with 25 wg of the drug per gram of body weight. The second injection was given 24 hr after the first one. 2
4
7
was considerably slower than in froglets. Thus a single injection of phenylhydrazine of 25 pglgm produced only about 75% hemolysis in these animals 7 days later. When phenylhydrazine was administered in serial injections, i.e., four doses of 12.5 pglgm body weight at l-day intervals, more than 60% hemolysis was produced 1 day after the last injection. Seven days after the beginning of treatment, 80% hemolysis was observed as shown in the third group in Table 1. The fourth group in Table 1 presents data on a smaller frog species, Rana pipiens. It is apparent that the hemolytic response to phenylhydrazine is faster in these animals than in the bullfrog, with 97% hemolysis observed in 7 days. Changes in RBC levels also reported in Table 1
correspond sults.
closely
to the percent
Hb re-
Erythropoiesis during Spontaneous Metamorphosis and in Adult Frogs The Hb regenerative phase was observed to be relatively slow in prometamorphic tadpoles which recovered their normal Hb values 2 or 3 months after treatment. Tadpoles, injected at the metamorphic climax, showed the fastest regeneration, with full recovery usually after the completion of metamorphosis, i.e., less than 1 month. Froglets recovered their Hb levels faster than prometamorphic tadpoles, i.e., between 1.5 and 2 months. Adult frogs, in which the slowest hemolytic response was observed, recovered fully to control values
410
DEVELOPMENTAL TABLE EFFECT
Group
1
OF PHENYLHYDRAZINE FR~CLETS
AND
BIOLOGY
ADULT
INJECTION FROGS”
RBC x 1000/mm3
Grams per 100 ml HB
R. catesbeiana froglets Control 5.5 * 0.5 1 day 0.15 f 0.02 2 days 0.06 i 0.03 3 days 0.04 * 0.02 7 days 0.10 zt 0.04 R. catesbeiam adults Control 5.0 ztz 0.6 1 day 3.0 zt 0.5 2 days 2.4 + 0.5 3 days 1.7 f 0.3 7 days 1.2 i 0.2 R. catesbeianu adults Control 5.0 +z 0.6 4 days 1.9 * 0.5 7 days 1.0 i 0.3 R. pipiens adults Control 7.2 * 0.4 4 days 1.7 i 0.6 7 days 0.25 + 0.05
ON
500 18 0.3 0.15 10
f 70 +5 * 0.1 * 0.1 i 1.5
510 230 170 100 80
* i * * i
90 60 45 30 10
510 * 90 100 * 20 50 * 10 680 i 40 93 * 44 30+ 7
n Except for the controls all the animals received intraperitoneal injection at the indicated dose. Ram catesbeiam froglets weighing between 5 and 10 gm, less than a week old after reaching stage XXV received a single ip injection of 25 fig/per gram body weight. Ram catesbeianu adults weighing between 306 and 400 gm were obtained from the Lemberger Co. (Oshkosh, Wisconsin). Group 2 received a single ip dose of 25 @g/per gram body weight. Group 3 received four doses of 12.5 fig/per gram body weight phenylhydrazine spaced at l-day intervals. Rana pipiens adults weighing between 30 and 70 gm were collected from local ponds. This group received four doses of 12.5 fig/per gram body weight spaced at l-day intervals. The Hb concentration was determined l-day, 2 days, etc., after initiation of treatment. The average of 5-10 samples for each experimental group (gram per 100 ml Hb) and + one standard deviation (SD) are given.
about ment.
1 month
after
initiation
of treat-
Effect of Triiodothyronine on the Hemolytic and Regenerative phases of Phenylhydmzine-Treated Tadpoles An increase in Hb concentration was observed when prometamorphic tadpoles were injected with T, (Fig. 2). The hormone changed the levels of Hb signifi-
VOLUME
27, 1972
cantly 8 days after injection of T,, 0.5 nmole/gm body weight, at which time hemoglobin levels of 3.90 (~0.3) and 5.40 (h0.4gm) gm/lOO ml were found, respectively. Eleven days after T,-treatment the values were 3.70 (hO.3) and 6.2 (+0.5) gm/lOO ml, respectively, as shown in the first and second columns of each group in Fig. 2. The third and fourth columns in Fig. 2 show the effect of intraperitoneal injection of phenylhydrazine 25 pglgm body weight, in normal and an equal total dose to the T,-treated tadpoles. When tadpoles were pretreated with T, and exposed to phenylhydrazine for 2 days, they showed a slower hemolytic response compared to controls. This particular trend is especially significant 8 and 11 days after treatment with T, with 30% and 20% hemolysis, respectively, while the control animals showed 50% and 55% hemolysis, respectively, 2 days after injection of a single dose of phenylhydrazine. The effect of T, treatment on erythropoiesis is shown in Fig. 3. It was mentioned earlier (Fig. 1 and Table 1) that phenylhydrazine produced a rapid hemolytic effect in amphibia, followed by a slow regenerative phase. Both effects are dependent on the developmental stage of the animal. Two weeks after administration of phenylhydrazine to pmmetamorphic tadpoles, there was less than 10% Hb relative to untreated controls. Administration of T, to phenylhydrazine-injected tadpoles produced an increase in Hb concentration which became significant 7 days later (Fig. 3). However, T, did not cause a significant increase in Hb concentration when given during the hemolytic phase and even 8-10 days after treatment with phenylhydrazine. Gel Electrophoresis of the Hemoglobins from Untreated and PhenylhydrazineTreated Amphibians An extensive shift in Hb patterns has
FLORES
AND
Hemolysis
FRIEDEN
and Anumn
411
Metamorphosis
7-
6-
5P 8 2 = 0
4-
3-
2-
l-
4
6 DAYS FIG. 2. Effect of phenylhydrazine in normal and pretreated tadpoles. The experimental groups consisted of Ranu catesbeiana tadpoles at stages XIII-XV maintained under experimental conditions described in the text. Blood for hemoglobin determination was withdrawn at indicated days after T, treatment. See Fig. 1 and text for other experimental details. [7, Control; g, intraperitoneal administration of T,, 0.5 nmole body weight at zero time;B, intraperitoneal injection of phenylhydrazone, 25 rg/g body weight, 2 days before withdrawal of blood for Hb determination. m, Combined pretreatment with T, and treatment with phenylhydrazine as described above. Doses of phenylhydrazine were adjusted to account for loss of body weight due to the hormonal treatment.
been shown to occur during amphibian metamorphosis (see review by Frieden and Just, 1970). In tadpoles immunized with adult frog Hb a different Hb appeared after completion of metamorphosis (Maniatis et al., 1969). It was of interest to compare the effect of phenylhydrazine on the Hb patterns obtained by polyacrylamide gel electrophoresis during both the hemolytic and regenerative phases. The main frog Hb component appeared at stage XXII. Treatment of tadpoles with phenylhydrazine at various stages of spontaneous metamorphosis leads to the appearance of this component as early as stage XX. When met,amorphosis was accelerated by administration of a low dose of T, (0.1 nmole/gm) an adult Hb pattern appeared after the front legs had come out (approximately stage XXI) even 36 days after hormone treatment. However when these animals were treated with
phenylhydrazine 19 and 20 days after receiving TS, they showed the major adult band as early as stage XVIII. When a larger dose (0.5 nmole/gm) of T, was used the tadpoles did not show any adult Hb components before their death, 10 days after T, treatment. It was also shown that the severe anemia induced by phenylhydrazine in all the experimental groups did not produce any atypical hemoglobins. We also found some naturally anemic frogs with an Hb concentration about 1 gm/lOO ml and having plasma highly colored with a green pigment, but their Hb pattern was qualitatively the same as that of the control frogs. Spectrophotometric Studies of the Interaction of Phenylhydrazine with Amphibian Blood The
nature
of the spectral
changes
412
DEVELOPMENTAL
BIOLOGY
VOLUME
27. 1972
2.0-
1.8 -
1.6 -
1.4 -
1.2 P 8
l.O-
2 ?i 0.8 -
0.6 -
* I DAYS
FIG. 3. Effect of triiodothyronine on the regenerative phase of phenylhydrazine-treated tadpoles. Tadpoles between stages XIII and XV were injected intraperitoneally with phenylhydrazine 25 pg/g body weight, at zero time and again 24 hrs later. Fourteen days from zero time, the animals were divided into two groups: the first group received no other treatment and was taken as a control group for erythropoiesis during regenerative phase; the second group received intraperitoneal injection of T, at 0.5 nmole/gm. Each figure in the abscissa represents days after hormonal treatment. 0, Phenylhydrazine; n , phenylhydrazine and T,.
which take place when either erythrocytes or free Hb are exposed to the action of electron transfer reagents have been thoroughly studied (Lemberg and Legge, 1949; Beaven and White, 1954; Mills and Randall, 1958; Jandl, 1963; et al., Kosower et al., 1965; Goldstein 1968). The spectral changes which take place when phenylhydrazine interacts with intact tadpole erythrocytes (in vivo and in vitro) and with hemolysates (in uitro) have been reported (Flores and Frieden, 1970). The results of this interaction between phenylhydrazine and the blood of metamorphosing tadpoles, T,-treated tadpoles, froglets, and adult
frogs are presented in Table 2. The progress of the reaction of phenylhydrazine with amphibian hemolysates was followed by recording the rate of decrease or increase of the absorbance at 410 and 630 nm, respectively. It was observed that frog hemolysate reacted considerably slower with phenylhydrazine than the hemolysates of the other experimental groups reported in Table 2. This incubation system permitted the exploration of the uptake of phenylhydrazine by amphibian erythmcytes and the interaction of the drug with red cell lysates. The uptake of phenylhydrazine by the erythrocytes of tadpoles through-
FLORES
AND
Hemolysis
FRIEDEN
and Anumn
TABLE EFFECT
Group
Sample
At 630 nm Prometamorphic tadpole Tadpole T, i.p.d Tadpole XXII-XXIII Froglet Adult
frog
Cells LYS Cells LYS Cells LYS Cells LYS Cells LYS
2
OF PHENYLHYDRAZINE
Time (min) for 50%) reaction completion” At 410 nm
0.5
3
0.5
3
0.5
3
0.5
3.5
1.5
9
ON AMPHIBIAN
Incubation phenylhydrazine
1.75 lo-‘M 6.2 7.4 6.5 8.0 6.3 8.0 5.7 6.4 4.0 5.1
x
+ * zt i +z f * zt * *
413
Metamorphosis
0.3 0.2 0.5 0.4 0.3 0.3 0.2 0.2 0.3 0.2
BLOOD
Injection system’ Phenylhydrazine
system” cont.
3.5 x lo-‘M 7.7 11.6 8.5 12.5 8.5 11.0 8.7 9.3 4.5 8.0
i i zt i * i i * i i
0.5 0.6 0.6 0.7 0.4 0.5 0.3 0.2 0.2 0.6
25 gml gm b.w.
50 pm/ gm b.w
7.4
* 0.3
7.8
f 0.4
8.9
+ 0.3
11.7
* 0.4
9.0
* 1.0
13.2
i 2.0
4.2
zt 0.3
6.2
+ 0.5
11.9
* 0.5
9.0 + 0.8
“The rate of reaction of hemolysate samples with phenylhydrazine was followed at 630 nm and 410 nm with a Beckman DB spectrophotometer equipped with an automatic recorder. The time in minutes for 50% completion of the reaction is reported here. The concentration of Hb was about 2 x 10m5 M and 2 x 10m6 M and that of phenylhydrazine was 1 x 10m3 M and 1 x lo- I M for the data at 630 nm and 410 nm, respectively. * Washed erythrocytes (Cells) of hemolysates (Lys) were incubated at 18” + 1°C for 5 min in 1.75 x 10 ’ M and 3.5 x lo- a M phenylhydrazine as indicated in Material and Methods. cIntraperitoneal injection of 25 pg and 50 pg phenylhydrazine per gram body weight (b.w.) was given to the experimental animals. One hour after injection the animals were sacrificed. Samples of blood were collected in deionized water and frozen immediately. Thereafter the samples were thawed and an equal volume of 2 x concentrated amphibian Ringer’s was added. The experimental figures reported in b and c represent spectral index defined as
Each value represents the average of 4-6 determinations * 1 standard d Tadpoles at stage XIII--XV received an intraperitoneal injection The experiments were performed 10 days after hormonal treatment.
out all metamorphic stages and T,treated animals is similar. However, erythrocytes of the adult frog show a poor penetration by phenylhydrazine as indicated by the low spectral index obtained by treatment of eryt,hrocytes with 0.175 and 0.35 mM phenylhydrazine, i.e., 4.0 h 0.3 and 4.5 * 0.2 respectively. This observation is consistent with data reported later on 3-O-methylglucose transport by amphibian erythrocytes, in which the erythrocytes of tadpoles throughout all metamorphic stages and froglets transported this nonmetabolizable sugar quite
deviation. of 0.3 nmole
T, per gram
body
weight.
efficiently while the adult frog erythrocytes appear to have lost this property. The interaction of phenylhydrazine with each hemolysate was greater with prometamorphic tadpoles and T,-treated animals than for the other groups reported here as indicated by a higher spectral index. The injection experiments were designed to study the correlation between the in vivo hemolytic effect and in vitro spectral changes produced by phenylhydrazine in amphibians, i.e., speed and severity of the hemolytic phase with
414
DEVELOPMENTAL
BIOLOGY
availability of the drug in the target red cell. With the highest dose of phenylhydrazine used, 50 pg (0.35 pmoles/gm body weight), the largest spectral change was observed in froglets, correlating with the fastest hemolytic effect produced by phenylhydrazine in these animals (Fig. 1 and Table 1). Both the T,-treated tadpoles and the adult frogs showed a small spectral change, also in agreement with the relatively weak in uivo hemolytic effect of phenylhydrazine in these groups (Fig. 2 and Table 1, respectively.) Value of Hemoglobin
to Tadpole
27, 1972
and Frieden, 1959). When tadpoles were made anemic by immersion in 35 PM phenylhydrazine at room temperature, they survived at 30°C comparable to nonanemic controls. However, when tadpoles were immersed in 2,4-dinitrophenol, greater mortality was observed after they were pretreated with 35 PM phenylhydrazine. This effect was noted at both 21°C and 30 “C (Fig. 4), but, as expected, at a lower dinitrophenol concentration at 30 “C. It should be recognized that the use of mortality as a criterion does not distinguish between a response to the greater need for oxygen or to reinforcement of the toxicity caused by the use of both drugs. Nevertheless, the data may be interpreted as indicating that in the tadpole stressed with 2,4-dinitrophenol, Hb and red cells do contribute survival value to the tadpole.
Survival
Since the first observation of the longevity of completely anemic tadpoles (Flores and Frieden, 1968), the question of their survival in relation to Hb was raised. Since Grass0 and Shephard (1968) reported that newts treated with acetylphenylhydrazine showed an oxygen consumption 64-67s of controls, we attempted to devise experiments which led to an increase in the oxygen requirement of the tadpole. Earlier we reported that tadpoles have a higher oxygen utilization at 30” and after immersion in or injection of 2,4-dinitrophenol (Lewis 0
VOLUME
Postmetamorphic Effects on Facilitated Diffusion in Anumn Erythrocytes A brief study was made of the effects of development on the facilitated diffusion of 3-O-methylglucose into the anuran erythrocyte. The rate of uptake
No
+NHNH,
n With
35/.1M
@NHNH2
T= 21”
10
15
20
T=30°
L
25
2,4-DINITROPHENOL
30
10
15
(MM)
FIG. 4. Groups of 10 Ram catesbeiana tadpoles, stages X-XV, were made anemic by immersion in pM phenylhydrazine for 48 hr at 21 + 1”. They were then immersed in the indicated concentrations of 2,4-dinitrophenol at 21°C and 3O”C, respectively, and mortality was followed over a g-day period. Data obtained by Somjai Sirivech, Department of Chemistry, Florida State University.
FLORES
AND FRIEDEN
Hemolysis
of radioactive 3-0-methylglucose was found to be similar using erythrocytes obtained from the following animals: (1) normal human, (2) R. grylio and R. catesbeianu tadpoles, with or without incubation in 5 PM T, for 4 hr, (3) R. catesbeiana tadpoles after injection of 0.3 nmole T, per gram for l-11 days, and (4) R. catesbeianu froglets. In contrast, the erythrocytes of three adult anuran species. R. catesbeiana, R. pipiens, and toads and turtle erythrocytes showed less than 5% of the rate of uptake of the other 4 groups reported above. These data indicate that the changes in the permeability of 3-O-methylglucose is a postmetamorphic response with no change during or soon after metamorphosis. In this regard, the tadpole and froglet cells behave as do fetal erythrocytes from certain other mammalian species, e.g., pigs, rabbits, guinea pigs, comparable to adult human sheep, erythrocytes. These mammals lose the capacity for sugar transport in their erythrocytes after birth (Widdas, 1955). DISCUSSION
Induction of Anemia in Amphibiu by Phenylhydmzine The results presented in this paper indicate the differential sensitivity of amphibians to the hemolytic action of phenylhydrazine and the facility of these species in developing severe anemia without serious impairment to their survival. The percent survival, which may be related to the oxygen demands at any particular stage in metamorphosis and in adulthood, showed the following decreasing order: froglets > prometamorphic tadpoles > adult frogs > metamorphic climax tad:poles > T,-treated tadpoles. The oxygen needs in T,-treated and metamorphic climax tadpoles are higher than in prometamorphic tadpoles, which is in agreement with the higher mortality
and Anuran
Metamorphosis
415
in the first two groups as compared with the last one. Furthermore, the severe anemia induced by phenylhydrazine in prometamorphic tadpoles make these animals more susceptible to the lethal effect of 2,4-dinitrophenol than the corresponding controls. The survival of highly anemic newts has been explained mainly on the basis of increased pulmonary oxygen intake (Grass0 and Shephard, 19681, while in hemoglobin-less R. pipiens frogs (Meints, 1969) and icefish (Hemmingsen and Douglas, 1970) the compensatory mechanism seems to be an increase in blood volume and higher cutaneous respiration. The higher mortality observed when the animals were exposed to 2,4-dinitrophenol, T,-treatment and metamorphic climax may be explained in terms of limiting oxygen availability (Grass0 and Shephard, 1968). Differential Sensitivity of Anuran Erythroc) tes to Phenylhydrazine Many factors may contribute to the differential sensitivity of amphibians to the hemolytic action of phenylhydrazine including the possible role of the skin and gills in the absorption of the drug administered by immersion to the metamorphosing tadpoles (Fig. 1). The liver, which shows changes in numerous enzyme activities (see review by Frieden and Just, 1970) as well as qualitative and quantitative changes in its subcellular particles (Bennett et al., 1970a, b) during metamorphosis or after treatment of the tadpole with thyroid hormones must also play an important role in the detoxication of the drug. The interaction of phenylhydrazine with the amphibian erythrocyte may be affected by the differences in permeability of the red cell membrane at different stages of metamophosis and different rates of reaction with Hb once the drug has penetrated. Thus both membrane and cytoplasmic effects must be con-
416
DEVELOPMENTAL
BIOLOGY
sidered. The low spectral index obtained when adult frog erythrocytes were incubated with the drug (Table 2) is in agreement with the lower hemolytic effect of phenylhydrazine in the adult frog (Table 1). The shift in Hb during metamorphosis would not explain, by itself, the different sensitivity of amphibians to phenylhydrazine, since froglets showing the adult frog Hb patterns were the most sensitive to the drug (Table 1). Two alternative explanations may be offered to account for these results. It has been observed that 3-O-methylglucose is efficiently transported into the red cells of both tadpoles and froglets, while this ability is lost in the erythrocytes of the adult frog. This could be also true for phenylhydrazine, thus accounting for the low spectral index when frog erythrocytes were incubated with the drug (Table 2). The other alternative would be that, although the Hb is the same for the froglets and the adult frog, other components of these erythrocytes may differ, e.g., organic phosphates, reduced glutathione. Organic phosphates play an important role in the interaction of Hb with oxygen (Benesch et al., 1968; Salhany et al., 1970). OxyHb participates in the oxidation of phenyldiimide (an intermediate in phenylhydrazine metabolism), leading to the formation of peroxyHb, which in the presence of glutathione gives rise to the formation of Fe(III)-Hb derivatives with subsequent production of free radicals, which may be harmful to the red cell (Kosower et al., 1965). The fact that the rates of reaction of phenylhydrazine (followed at 410 nm and 630 nm) with lysates of tadpole and froglet erythrocytes differ considerably from that of the frog erythrocyte lysates supports this idea. Erythropoiesis
in Amphibia
Maniatis and Ingram (1971a) have recently shown that the liver is the organ where the erythroid cell matures both
VOLUME
27. 1972
before and during metamorphosis of R. catesbeianu tadpoles. The corresponding site in the frog is presumed to be the bone marrow. The rate of regeneration of Hb observed in this work was as follows: climax tadpoles > T,-treated tadpoles > adult frog > froglet > prometamorphic tadpoles is consistent with the delay in the onset of erythropoiesis (13-32 days) after acetylphenylhydrazine reported by Grass0 and Shephard (1968) in anemic newts. However, in the adult frog (Rana pipiens), erythropoiesis starts soon after the induced hemolytic phase (Meints, 1969). Although phenylhydrazine has toxic side effects in kidney and liver (Schuckmann, 1969), it may not damage other erythropoietic for the higher organs, thus accounting regenerative rate observed in froglets and adult frogs. If there is a shift in erythropoietic organs with active replacement of tadpole erythrocytes by the new frog erythrocyte line, the highest rate of regeneration would be expected for the tadpole at its metamorphic climax, which is consistent with our findings. The faster regeneration observed in the T,-treated tadpoles can be accounted for either by the loss of water from the intravascular compartment or true erythropoietic stimulation by thyroid hormones (Gallagher and Ford, 1969). It is interesting to note that the more time allowed between phenylhydrazine and T, administration, the higher the Hb concentration in those animals receiving hormonal treatment. This observation also supports the idea that reversible damage at the site of erythropoiesis plays a major role in determining the rate of regeneration. Effect of Phenylhydnxine globin Patterns
on Hemo-
The results reported in this paper indicate that the shift in Hb patterns which occur during metamorphosis is dependent only on the developmental
FLORES
.~ND FRIEDEN
Hem013 Isis and Anumn
stage of metamorphosing tadpoles. Contrary to what has been demonstrated mainly for some mammalian species, the severe anemia induced by phenylhydrazine did not produce any shift or preferential appearance of either fetal type (tadpole Hb) or abnormal Hb in the experimental animals studied in this work. However, phenylhydrazine treatment proved to be useful in showing the appearance of adult Hb patterns earlier than in appropriate controls. The appearance of adult Hb patterns (using phenylhydrazine) as early as stage XVIII in those tadpoles treated with a low dose of T, may be explained in terms of stimulation of spleen erythropoiesis as a consequence of anemia. It has been shown that in mammals, hypoxia (CO) stimulates spleen erythropoiesis (Ramsey, 1969). Since the bone starts developing at stage XX (Kemp and Hoyt, 1969), the earlier appearance of adult Hb at stage XVIII would also suggest the role of the spleen in producing frog Hb at this early stage in metamorphosis. This research was supported by U. S. Public Health Service Grant HD 01236 from the National Institutes of Child Health and Human Development. This is paper No. 44 in a series from this laboratory on the biochemistry of amphibian metamorphosis. REFERENCES BANERJEE, V., and BANERJEE, M. (1969). Seasonal variations of erythrocyte number and hemoglobin content in a common Indian lizard Vunanus monitor. 2001 Anz. 182, 203-207. BEAVEN, G. H., and WHITE, J. C. (1954). Oxydation of phenylhydrazine in the presence of oxyhemoglobin and the origin of Heinz bodies in erythrocytes. Nature (London) 173, 389-391. BENBASSAT, J. (1970). Erythroid cell development during natural amphibian metamorphosis. Deuelop. Biol. 21, 557-583. BENESCH, R., BENESCH, R. E., and Yu, C. I. (1968). Reciprocal binding of oxygen and diphosphoglycerate by human hemoglobin. Proc. Nat. Acad. Sci. U.S. 59, 526-532. BENNETT, T. P., GLENN, J. S., and SHELDON, H. (1970a). Changes in the fine structure of tadpole (R. catesbeianu) liver during thyroxine-induced metamorphosis. Deuelop. Biol. 22, 232-248.
Metamorphosis
417
BENNETT, T. P., and GLENN, J. S. (197Ob). Fine structural changes in liver cells of R. catesbeiana during natural metamorphosis. Deuelop. Biol. 22, 535-560. CAMERON, J. N., and WOHLSCHLAG, D. E. (1969). Respiratory response to experimentally induced anemia in the pinfish (Lagodon rhomboides). J. Exp. Biol. 50, 307-317. DEGRAAF, A. R. (1957). A note on the oxygen requirements of Xenopus laeuis. J. Exp. Biol. 34, 173-176. DEWITT, W. (1968). Microcytic response to thyroxine administration. J. Mol. Viol. 32, 502-504. EWER, D. W. (1959). A toad (Xenopus laeuis) without haemoglobin. Nature (London) 183, 271. FLORES, G. (1969). Hemolytic and spectral effects of phenylhydrazine and hydrazine derivatives in amphibian red cells. M. S. Thesis. The Florida State University, Tallahassee, Florida. FLORES, G., and FRIEDEN, E. (1968). Induction .and survival of hemoglobin-less and erythrocyte-less tadpoles and young bullfrogs. Science 151, 101-103. FLORES, G., and FRIEDEN, E. (1970). Structural requirements for the hemolytic effect of phenylhydrazine derivatives on amphibian red cells. J. Pharmacol. Exp. Ther. 174, 463-472. FRIEDEN, E., and JUST, J. J. (1970). Hormonal responses in Amphibian metamorphosis. In “Biochemical Actions of Hormones” (E. G. Litwack, ed), pp. l-52. Academic Press, New York. FRIEDMAN, G. B., ALGARD, F. T., and MCCURDY H. M. (1969). Determination of red blood cell count and hemoglobin content of urodele blood. Anut. Rec.163, 55-57. GALLAGHER, N. I., and FORD, G. H. (1969). Effect of hyperoxia on the erythropoietic response of sodium L-triiodothyronine. Proc. Sot. Exp. Biol. Med. 130, 672-674. FOLDSTEIN, G. W., HAMANAKER, L., and SCHMID, R. (1968). The catabolism of Heinz bodies: An experimental model demonstrating conversion to non-bilinium catabolites. Blood 31, 388-395. GRASSO, J. A., and SHEPHARD, D. C. (1968). Experimental production of totally anemic newts. Nature (London) 218, 1274-1276. HEMMINGSEN, E. A., and DOUGLAS, E. L. (1970). Respiratory characteristics of the hemoglobinfree fish (Chaemocephalus acemtus). Comp. Biochem. Physiol. 33, 733-744. HERNER, A. E., and FRIEDEN, E. (1961). Biochemical changes during anuran metamorphosis. VIII. Changes in the nature of red cell proteins. Arch. Biochem. Biophys. 95, 25-35. HOLLYFIELD, J. G. (1966). Erythrocyte replacement at metamorphosis in the frog, R. pipiens. J. Morphol. 119, 1-6. JANDL, J. H. (1963). The Heinz body hemolytic anemias. Ann. Intern. Med. 58, 702-709.
418
DEVELOPMENTAL
BIOLOGY
KEMP, N. E., and HOYT, J. A. (1969). Sequence of ossification in the skeleton of growing and metamorphosing tadpoles of Rana pipiens. J. Morphol. 129, 415-443. KOSOWER, N. S., VANDERHOFF, G. A., KOSOWER, E. M., and HUANG, P. C. (1965). Decreased glutathione content of human erythrocytes produced by methyl phenylazoformate. Biochem. Biophys. Res. Commun. 20, 469-474. LEMBERG, R., and LEGGE, J. W. (1949). “Hematin Compounds and Bile Pigments: Their Constitution, Metabolism, and Function,” pp. 524-528. Wiley (Interscience) New York. LEWIS, E. J., and FRIEDEN, E. (1959). Biochemistry of amphibian metamorphosis: Effect of triiodothyronine, thyroxin, and dinitrophenol on the respiration of the tadpole. Endocn’nology 65, 273-282. MANIATIS, G. M., and INGRAM, V. M. (1971a). Erythropoiesis during amphibian metamorphosis. I. Site of maturation of erythrocytes in Rano catesbeiam. J. Cell Biol. 49, 372-379. MANIATIS, G. M., and INGRAM, V. M. (1971b). Erythropoiesis during amphibian metamorphosis. II. Immunochemical study of larval and adult hemoglobins of Rana catesbeianu. J. Cell Biol. 49, 380-389. MANIATIS, G. M., and INGRAM, V. M. (1971c). Erythropoiesis during amphibian metamorphosis. III. Immunochemical detection of tadpole and frog hemoglobins (Rana catesbeiuna) in single erythrocytes. J. Cell Biol. 49, 390-404. MANIATIS, G. M., STEINER, L. S., and INGRAM, V. M. (1969). Tadpole antibodies versus frog hemoglobin and their effect on development. Science 165, 67-69. MEINTS, R. (1969). Erythropoietic activity in Ranu pipiens. The influence of severe hemolytic induced anemia on spleen and peripheral blood activities. Comp. Biochem. Physiol. 30, 383-389. MILLS, G. C., and RANDALL, H. P. (1958). Hemo-
VOLUME
27. I972
globin catabolism. II. The protection of hemoglobin from oxidative breakdown in the intact erythrocyte. J. Biol. Chem. 232, 589-598. Moss, B., and INGRAM, V. M. (1968a). Hemoglobin synthesis during amphibian metamorphosis. I. Chemical studies on the hemoglobins from the larval and adult stages of Ranu catesbeiana J. Mol. Biol. 32, 481-492. Moss, B., and INGRAM, V. M. (1968). Hemoglobin synthesis during amphibian metamorphosis. II. Synthesis of adult hemoglobin following thyroxine administration. J. Mol. Biol. 32, 493-504. RAMSEY, J. M. (1969). Immediate hematological response to the rat to exposure to CO. J. Physiol. (London) 202, 297-304. RUDD, J. R. (1954). Vertebrates without erythrocytes and blood pigments. Nature (London) 173, 848-850. RUGH, R. (1962). “Experimental Embryology” p. 50. Burgess, Minneapolis, Minnesota. SALHANY, J. M. ELIOT, R. S., and MIZUKAMI, H. (1970). Effect of 2,3-diphosphoglycerate on the kinetics of deoxygenation of human hemoglobin. Biochem. Biophys. Res. Commun. 39, 1052. SCHUCKMANN, F. (1969). Different forms of phenylhydrazine intoxication. Zentrolbl. Argeitsmed. Arbeitsschutz 19, 338-341. SULLIVAN, B., and RIGGS, A. (1964). Haemoglobin: Reversal of oxidation and polymerization in turtle red cells Nature (London) 204, 1098-1099. TAYLOR, A., and KOLLROS, J. J. (1946). Stages in normal development of Ranu pipiens larvae. Anat. Rec. 94, 7-23. THEIL, E. C. (1967). The synthesis of hemoglobin and non-hemoglobin protein of amphibian red blood cells related to metamorphosis. Biochim. Biophys. Acta 138, 175-185. WIDDAS, W. F. (1955). Hexose permeability of foetal erythrocytes. J. Physiol. (London) 127, 318-327.
BIOLOGY
Hemolytic
27, 406418
(1972)
Effect of Phenylhydrazine Metamorphosis GUSTAVO
Department
of Chemistry,
during
Amphibian
FLORES ’ AND EARL FRIEDEN* Florida Accepted
State
Uniuersity,
November
Tallahassee,
Florida
32306
9, 1971
The correlation between the spectral changes in hemoglobin and the severity of anemia induced by phenylhydrazine treatment was studied for the differential sensitivity of amphibians to the drug. Froglets were the most sensitive to phenylhydrazine, follrwed by prometamorphic tadpoles, adult frogs, metamorphic climax tadpoles, and triiodothyronine-treated tadpoles. The different sensitivities to the hemolytic action of the drug in these animals was rationalized in terms of accessibility, uptake, and detoxication of phenylhydrazine, and a different rate of removal of damaged cells. Postmetamorphic responses were noted for the low uptake of phenylhydrazine by erythrocytes and the loss of facilitated diffusion of 3-0-methylglucose by the erythrocytes of the adult frog. The rate of regeneration of erythrocytes (erythropoiesis) after phenylhydrazine treatment followed an order almost opposite to that observed in the hemolytic process. Thus, tadpoles at metamorphic climax showed the maximum rate of regeneration, followed by T,-treated tadpoles, adult frogs, froglets, and prometamorphic tadpoles. Erythropoiesis immediately following the hemolytic phase did not reveal any abnormal hemoglobins. Survival after exposure to phenylhydrazine followed the same order described for hemolytic susceptibility, i.e., froglets had the highest percent survival. Tadpoles immersed in 2,4-dinitrophenol were more sensitive to the lethal effects of phenylhydrazine.
box turtle with more than 90% of its Hb Vertebrate species completely lacking present as methemoglobin has also been erythrocytes and hemoglobin (Hb) have reported (Sullivan and Riggs, 1964). Cerbeen reported (Rudd, 1954). The main tain amphibians and other species are respiratory compensation in a family of characterized by seasonal changes in Hb fishes (Chaemichthyidae) appears to be concentration which may be adaptive (Anan increase of blood volume and high cu- drew, 1965; Theil, 1967; Friedman et aZ., taneous respiration (Hemmingsen and 1969; Banerjee and Banerjee, 1969). We have reported the unusual sensitivDouglas, 1970). Occasionally, amphibians ity of amphibian red blood cells to phenylwithout erythrocytes have been found hydrazine and other hydrazine derivatives (DeGraaf, 1957; Ewer, 1959). Nate (per(Flores and Frieden, 1968, 1970). A low sonal communication) has found mutant dose of phenylhydrazine eliminates the tadpoles which are completely lacking in erythrocytes and Hb of Ranu tadpoles in Hb. This has been confirmed in our laboraa short time, and the animals survive withtory with observations that some tadpoles out obvious difficulty. A direct correlation and adult bullfrogs survived with blood Hb levels as low as 1.0 gm/lOO ml (Flores, between the spectral changes of the Hb 1969). The survival of a specimen of the molecule observed in vitro and the in vivo effect of hemolytically active hydrazines 1 Present address: Oregon State University, Enwas also demonstrated (Flores and Frievironmental Health Science Center, Corvallis, den, 1970). The experimental production Oregon 97331. of anemic newts (Grass0 and Shephard, * Reprint requests should be sent to: Earl Frieden, 1968), anemic frogs (Meints, 1969) and Department of Chemistry, Florida State University, Tallahassee, Florida 32306. anemic fish (Cameron and Wohlschlag, INTRODUCTION
Copyright
0 1972 by Academic
Press, Inc.
406
FLORES
AND FRIEDEN
Hemolysis
1969) have also been reported. The numerous biochemical changes that take place during amphibian metamorphosis and their adaptive implications have been reviewed most recently by Frieden and Just (1970). Prominent among those changes is the shift in the main erythropoietic organ of the larval tadpole as compared to the adult frog (Maniatis and Ingram, 1971a-c). This shift appears to result in the production of a new adult red blood cell line differing from the larval erythrocyte in several characteristics including hemoglobin patterns, osmotic fragility, size and shape of the erythrocytes (Herner and Frieden, 1961; Hollyfield, 1966; Moss and Ingram, 1968a, b; Dewitt, 1968; Benbassat, 1970; Maniatis and Ingram, 1971a-c). Therefore we decided to explore the susceptibility of amphibian erythrocytes to the hemolytic action of phenylhydrazine (1) during spontaneous metamorphosis, (2) in the triiodothyronine-(T,)treated tadpole, and (3) in the adult frog. We also sought to correlate any differential susceptibility to phenylhydrazine with the developmental stage of the animal and to study the regenerative phase (erythropoiesis) taking place immediately after the hemolytic phase in phenylhydrazine-treated amphibians and identify their Hb patterns during recovery. METHODS
AND
MATERIALS
Phenylhydrazine hydrochloride was obtained from the Fischer Scientific Co., St. Louis, Missouri, and was recrystallized from concentrated hydrochloric acid every 3-4 months. The crystals were kept in a desiccator at -15°C to minimize oxidation. L-Triiodothyronine (T,) was donated by Smith, Kline, and French Labs., Philadelphia, Pennsylvania. Cyanmethemoglobin reagent and standard were purchased from Hycel, Inc., Houston, Texas. The chemicals for polyacrylamide gel electrophoresis were obtained from Eastman Organic Chemicals, Roch-
and Anumn
Metamorphosis
407
ester, New York. 3-O-methy114C-n-glucase was purchased from New England Nuclear, Boston, Massachusetts. Rana catesbeiana tadpoles and frogs were purchased from the Lemberger Co., Oshkosh, Wisconsin. Froglets were selected from R. catesbeiana tadpoles which had completed metamorphosis within 1 month of their use. R. grylio and R. pipiens were collected from local ponds. All animals were starved at least 1 day before and during the experiment. The laboratory temperature was held at 22 + 2°C. The metamorphic development of bullfrog tadpoles was classified according to the stage criteria of Taylor and Kollros (1946). Prometamorphosis refers to stages X to XX and metamorphic climax to stages XX to XXV (for further details see review of Frieden and Just, 1970). Blood was obtained by exposing the heart of the tadpole and froglets, removing any tissue fluid and cutting or piercing the arterial trunk and allowing the blood to flow into a heparinized pipette. For adult frogs, the samples were obtained by heart puncture using a 5-ml syringe fitted with a 20-gauge needle. The concentration of Hb was determined by the cyanmethemoglobin method as described earlier (Flores and Frieden, 1970). Individual samples of blood were collected in heparinized (0.01%) amphibian Ringer’s (Rugh, 1962). These samples were diluted as necessary and the erythrocytes counted in a hemacytometer (Neubauer by C. A. Hauser and Sons, Philadelphia). The separation of the hemoglobins by polyacrylamide gel disc electrophoresis using lysates of amphibian erythrocytes was performed as described by Moss and Ingram (1968a). Experimental design. The in vivo treatment consisted of an intraperitoneal injection of 35 mM phenylhydrazine (1 or 2 injections of 5 &gm) or immersion of the animals in 35 PM phenylhydrazine in tap water, 6 animals per liter. All
408
DEVELOPMENTAL
BIOLOGY
phenylhydrazine solutions were brought to pH 7.0-7.4 with dilute NaOH. T, was injected intraperitoneally-0.1 nmoles or 0.5 nmole per gram of tadpole. For the in vitro incubation, individual samples of frog blood or pooled samples (3 to 10) of tadpole or froglet blood were collected in heparinized (0.01% heparin) amphibian Ringer’s solution (Rugh, 1948) and washed 3 times with amphibian Ringers. A 1: 100 suspension of erythrocytes in amphibian Ringer’s was incubated for 5 min at 18 + 1°C with the indicated concentration of phenylhydrazine. The cells were centrifuged at maximum speed in a clinical centrifuge for 1 min and washed with 30 volumes of amphibian Ringers. The cells were lysed in one-half amphibian Ringer’s by freezing and thawing 3 times. Another aliquot of erythrocytes was lysed before incubation with phenylhydrazine. All the samples were adjusted to a final composition of amphibian Ringer’s and centrifuged for 10 min and the clear supernatant was taken for spectral analysis using a Beckman DB spectrophotometer. RESULTS
Hemolytic Effect of Phenylhydrazine during Spontaneous metamorphosis Figure 1 shows the effect of phenylhydrazine administered by immersion or intraperitoneal injection to tadpoles at various stages of metamorphosis. The immersion data, Fig. lA, indicate that the more advanced the metamorphosis the less sensitive were the tadpoles to the hemolytic action of the drug. Thus, 4 days after initiation of treatment all groups except the froglets (stage XXV) showed a minimum Hb concentration, with 98-99% hemolysis in tadpoles between stages X and XIX, over 97% hemolysis in the group at stages XX and XXI, and 90% hemolysis in animals between stages XXII and XXIV. Froglets were relatively insensitive to the immersion treatment as indicated by the
VOLUME
21, 1972
fact that less than 50% hemolysis was produced 2 days after immersion of the experimental group in 35 &f phenylhydrazine. The decreasing hemolytic effect of the drug while metamorphosis proceeds may be due to the decreased availability of phenylhydrazine to the blood and other tissues because of thickening of the skin and loss of the contribution of the gills to the absorption of the drug (Frieden and Just, 1970). The effect of intraperitoneal injection of phenylhydrazine in tadpoles at various stages of spontaneous metamorphosis is shown in Fig. 1B. Two injections of 25 pg (0.175 pmole) of phenylhydrazine per gram body weight were given to the animals, with an interval of 24 hr after the first injection. The maximum hemolytic effect is reached 4 days after the first injection. A fast hemolytic phase (represented by experimental groups 2 and 4 days after administration of the drug) is followed by a slow red cell regenerative phase (represented by the group 7 days after initiation of the experiments). Thus with the exception of froglets for which the highest hemolytic effect of the drug is observed, hemolysis is greater at the lower metamorphic stages, while the regenerative phase is faster with more advanced metamorphosis. Effect of Phenylhydrazine Injection Froglets and Adult Frogs
in
Table 1 shows the effect of intraperitoneal injection of phenylhydrazine into young froglets and in two different adult species. As in Fig. 1, the injection of phenylhydrazine proved to be more effective in producing a fast hemolytic response in froglets than it did in tadpoles. Concentrations of Hb as low as 3% relative to controls were obtained as early as 1 day after injection of phenylhydrazine. In the adult bullfrog (second and third groups in Table l), the hemolytic process
FLORES
f 8 2 w 0
AND FRIEDEN
Hemolysis
and Anumn
409
Metamorphosis
0.8 -
0.6-
7 2 4 DAYS FIG. 1. Effect of immersion and intraperitoneal injection of phenylhydrazine during spontaneous metamorphosis. Each experimental value (vertical bars) represents the average of 7-10 animals. Vertical lines indicate = one standard deviation from average. 0, Stages X-XIII (4.0 + 0.5); N, stages XVIII and XIX (4.5 + 0.4); 0, stages XX and XXI (6.4 i 0.6); I, stages XXII-XXIV (7.2 + 0.8);a, stages XXV (5.5 + 0.5). The grams per 100 ml (gram 9%) Hb for the controls of each experimental group are given in parentheses and represent the average of 7-10 analyses with standard deviation. Left, Fig. 1A: Immersion of tadpoles in 35 FM phenylhydrazine solution. Six animals per liter of solution were immersed for 24 hrs at room temperature (23 + 1°C). Samples were collected 2, 4, and 7 days after initiation of experiment. Right, Fig. 1B: Intraperitoneal injection of phenylhydrazine. The tadpoles were injected twice with 25 wg of the drug per gram of body weight. The second injection was given 24 hr after the first one. 2
4
7
was considerably slower than in froglets. Thus a single injection of phenylhydrazine of 25 pglgm produced only about 75% hemolysis in these animals 7 days later. When phenylhydrazine was administered in serial injections, i.e., four doses of 12.5 pglgm body weight at l-day intervals, more than 60% hemolysis was produced 1 day after the last injection. Seven days after the beginning of treatment, 80% hemolysis was observed as shown in the third group in Table 1. The fourth group in Table 1 presents data on a smaller frog species, Rana pipiens. It is apparent that the hemolytic response to phenylhydrazine is faster in these animals than in the bullfrog, with 97% hemolysis observed in 7 days. Changes in RBC levels also reported in Table 1
correspond sults.
closely
to the percent
Hb re-
Erythropoiesis during Spontaneous Metamorphosis and in Adult Frogs The Hb regenerative phase was observed to be relatively slow in prometamorphic tadpoles which recovered their normal Hb values 2 or 3 months after treatment. Tadpoles, injected at the metamorphic climax, showed the fastest regeneration, with full recovery usually after the completion of metamorphosis, i.e., less than 1 month. Froglets recovered their Hb levels faster than prometamorphic tadpoles, i.e., between 1.5 and 2 months. Adult frogs, in which the slowest hemolytic response was observed, recovered fully to control values
410
DEVELOPMENTAL TABLE EFFECT
Group
1
OF PHENYLHYDRAZINE FR~CLETS
AND
BIOLOGY
ADULT
INJECTION FROGS”
RBC x 1000/mm3
Grams per 100 ml HB
R. catesbeiana froglets Control 5.5 * 0.5 1 day 0.15 f 0.02 2 days 0.06 i 0.03 3 days 0.04 * 0.02 7 days 0.10 zt 0.04 R. catesbeiam adults Control 5.0 ztz 0.6 1 day 3.0 zt 0.5 2 days 2.4 + 0.5 3 days 1.7 f 0.3 7 days 1.2 i 0.2 R. catesbeianu adults Control 5.0 +z 0.6 4 days 1.9 * 0.5 7 days 1.0 i 0.3 R. pipiens adults Control 7.2 * 0.4 4 days 1.7 i 0.6 7 days 0.25 + 0.05
ON
500 18 0.3 0.15 10
f 70 +5 * 0.1 * 0.1 i 1.5
510 230 170 100 80
* i * * i
90 60 45 30 10
510 * 90 100 * 20 50 * 10 680 i 40 93 * 44 30+ 7
n Except for the controls all the animals received intraperitoneal injection at the indicated dose. Ram catesbeiam froglets weighing between 5 and 10 gm, less than a week old after reaching stage XXV received a single ip injection of 25 fig/per gram body weight. Ram catesbeianu adults weighing between 306 and 400 gm were obtained from the Lemberger Co. (Oshkosh, Wisconsin). Group 2 received a single ip dose of 25 @g/per gram body weight. Group 3 received four doses of 12.5 fig/per gram body weight phenylhydrazine spaced at l-day intervals. Rana pipiens adults weighing between 30 and 70 gm were collected from local ponds. This group received four doses of 12.5 fig/per gram body weight spaced at l-day intervals. The Hb concentration was determined l-day, 2 days, etc., after initiation of treatment. The average of 5-10 samples for each experimental group (gram per 100 ml Hb) and + one standard deviation (SD) are given.
about ment.
1 month
after
initiation
of treat-
Effect of Triiodothyronine on the Hemolytic and Regenerative phases of Phenylhydmzine-Treated Tadpoles An increase in Hb concentration was observed when prometamorphic tadpoles were injected with T, (Fig. 2). The hormone changed the levels of Hb signifi-
VOLUME
27, 1972
cantly 8 days after injection of T,, 0.5 nmole/gm body weight, at which time hemoglobin levels of 3.90 (~0.3) and 5.40 (h0.4gm) gm/lOO ml were found, respectively. Eleven days after T,-treatment the values were 3.70 (hO.3) and 6.2 (+0.5) gm/lOO ml, respectively, as shown in the first and second columns of each group in Fig. 2. The third and fourth columns in Fig. 2 show the effect of intraperitoneal injection of phenylhydrazine 25 pglgm body weight, in normal and an equal total dose to the T,-treated tadpoles. When tadpoles were pretreated with T, and exposed to phenylhydrazine for 2 days, they showed a slower hemolytic response compared to controls. This particular trend is especially significant 8 and 11 days after treatment with T, with 30% and 20% hemolysis, respectively, while the control animals showed 50% and 55% hemolysis, respectively, 2 days after injection of a single dose of phenylhydrazine. The effect of T, treatment on erythropoiesis is shown in Fig. 3. It was mentioned earlier (Fig. 1 and Table 1) that phenylhydrazine produced a rapid hemolytic effect in amphibia, followed by a slow regenerative phase. Both effects are dependent on the developmental stage of the animal. Two weeks after administration of phenylhydrazine to pmmetamorphic tadpoles, there was less than 10% Hb relative to untreated controls. Administration of T, to phenylhydrazine-injected tadpoles produced an increase in Hb concentration which became significant 7 days later (Fig. 3). However, T, did not cause a significant increase in Hb concentration when given during the hemolytic phase and even 8-10 days after treatment with phenylhydrazine. Gel Electrophoresis of the Hemoglobins from Untreated and PhenylhydrazineTreated Amphibians An extensive shift in Hb patterns has
FLORES
AND
Hemolysis
FRIEDEN
and Anumn
411
Metamorphosis
7-
6-
5P 8 2 = 0
4-
3-
2-
l-
4
6 DAYS FIG. 2. Effect of phenylhydrazine in normal and pretreated tadpoles. The experimental groups consisted of Ranu catesbeiana tadpoles at stages XIII-XV maintained under experimental conditions described in the text. Blood for hemoglobin determination was withdrawn at indicated days after T, treatment. See Fig. 1 and text for other experimental details. [7, Control; g, intraperitoneal administration of T,, 0.5 nmole body weight at zero time;B, intraperitoneal injection of phenylhydrazone, 25 rg/g body weight, 2 days before withdrawal of blood for Hb determination. m, Combined pretreatment with T, and treatment with phenylhydrazine as described above. Doses of phenylhydrazine were adjusted to account for loss of body weight due to the hormonal treatment.
been shown to occur during amphibian metamorphosis (see review by Frieden and Just, 1970). In tadpoles immunized with adult frog Hb a different Hb appeared after completion of metamorphosis (Maniatis et al., 1969). It was of interest to compare the effect of phenylhydrazine on the Hb patterns obtained by polyacrylamide gel electrophoresis during both the hemolytic and regenerative phases. The main frog Hb component appeared at stage XXII. Treatment of tadpoles with phenylhydrazine at various stages of spontaneous metamorphosis leads to the appearance of this component as early as stage XX. When met,amorphosis was accelerated by administration of a low dose of T, (0.1 nmole/gm) an adult Hb pattern appeared after the front legs had come out (approximately stage XXI) even 36 days after hormone treatment. However when these animals were treated with
phenylhydrazine 19 and 20 days after receiving TS, they showed the major adult band as early as stage XVIII. When a larger dose (0.5 nmole/gm) of T, was used the tadpoles did not show any adult Hb components before their death, 10 days after T, treatment. It was also shown that the severe anemia induced by phenylhydrazine in all the experimental groups did not produce any atypical hemoglobins. We also found some naturally anemic frogs with an Hb concentration about 1 gm/lOO ml and having plasma highly colored with a green pigment, but their Hb pattern was qualitatively the same as that of the control frogs. Spectrophotometric Studies of the Interaction of Phenylhydrazine with Amphibian Blood The
nature
of the spectral
changes
412
DEVELOPMENTAL
BIOLOGY
VOLUME
27. 1972
2.0-
1.8 -
1.6 -
1.4 -
1.2 P 8
l.O-
2 ?i 0.8 -
0.6 -
* I DAYS
FIG. 3. Effect of triiodothyronine on the regenerative phase of phenylhydrazine-treated tadpoles. Tadpoles between stages XIII and XV were injected intraperitoneally with phenylhydrazine 25 pg/g body weight, at zero time and again 24 hrs later. Fourteen days from zero time, the animals were divided into two groups: the first group received no other treatment and was taken as a control group for erythropoiesis during regenerative phase; the second group received intraperitoneal injection of T, at 0.5 nmole/gm. Each figure in the abscissa represents days after hormonal treatment. 0, Phenylhydrazine; n , phenylhydrazine and T,.
which take place when either erythrocytes or free Hb are exposed to the action of electron transfer reagents have been thoroughly studied (Lemberg and Legge, 1949; Beaven and White, 1954; Mills and Randall, 1958; Jandl, 1963; et al., Kosower et al., 1965; Goldstein 1968). The spectral changes which take place when phenylhydrazine interacts with intact tadpole erythrocytes (in vivo and in vitro) and with hemolysates (in uitro) have been reported (Flores and Frieden, 1970). The results of this interaction between phenylhydrazine and the blood of metamorphosing tadpoles, T,-treated tadpoles, froglets, and adult
frogs are presented in Table 2. The progress of the reaction of phenylhydrazine with amphibian hemolysates was followed by recording the rate of decrease or increase of the absorbance at 410 and 630 nm, respectively. It was observed that frog hemolysate reacted considerably slower with phenylhydrazine than the hemolysates of the other experimental groups reported in Table 2. This incubation system permitted the exploration of the uptake of phenylhydrazine by amphibian erythmcytes and the interaction of the drug with red cell lysates. The uptake of phenylhydrazine by the erythrocytes of tadpoles through-
FLORES
AND
Hemolysis
FRIEDEN
and Anumn
TABLE EFFECT
Group
Sample
At 630 nm Prometamorphic tadpole Tadpole T, i.p.d Tadpole XXII-XXIII Froglet Adult
frog
Cells LYS Cells LYS Cells LYS Cells LYS Cells LYS
2
OF PHENYLHYDRAZINE
Time (min) for 50%) reaction completion” At 410 nm
0.5
3
0.5
3
0.5
3
0.5
3.5
1.5
9
ON AMPHIBIAN
Incubation phenylhydrazine
1.75 lo-‘M 6.2 7.4 6.5 8.0 6.3 8.0 5.7 6.4 4.0 5.1
x
+ * zt i +z f * zt * *
413
Metamorphosis
0.3 0.2 0.5 0.4 0.3 0.3 0.2 0.2 0.3 0.2
BLOOD
Injection system’ Phenylhydrazine
system” cont.
3.5 x lo-‘M 7.7 11.6 8.5 12.5 8.5 11.0 8.7 9.3 4.5 8.0
i i zt i * i i * i i
0.5 0.6 0.6 0.7 0.4 0.5 0.3 0.2 0.2 0.6
25 gml gm b.w.
50 pm/ gm b.w
7.4
* 0.3
7.8
f 0.4
8.9
+ 0.3
11.7
* 0.4
9.0
* 1.0
13.2
i 2.0
4.2
zt 0.3
6.2
+ 0.5
11.9
* 0.5
9.0 + 0.8
“The rate of reaction of hemolysate samples with phenylhydrazine was followed at 630 nm and 410 nm with a Beckman DB spectrophotometer equipped with an automatic recorder. The time in minutes for 50% completion of the reaction is reported here. The concentration of Hb was about 2 x 10m5 M and 2 x 10m6 M and that of phenylhydrazine was 1 x 10m3 M and 1 x lo- I M for the data at 630 nm and 410 nm, respectively. * Washed erythrocytes (Cells) of hemolysates (Lys) were incubated at 18” + 1°C for 5 min in 1.75 x 10 ’ M and 3.5 x lo- a M phenylhydrazine as indicated in Material and Methods. cIntraperitoneal injection of 25 pg and 50 pg phenylhydrazine per gram body weight (b.w.) was given to the experimental animals. One hour after injection the animals were sacrificed. Samples of blood were collected in deionized water and frozen immediately. Thereafter the samples were thawed and an equal volume of 2 x concentrated amphibian Ringer’s was added. The experimental figures reported in b and c represent spectral index defined as
Each value represents the average of 4-6 determinations * 1 standard d Tadpoles at stage XIII--XV received an intraperitoneal injection The experiments were performed 10 days after hormonal treatment.
out all metamorphic stages and T,treated animals is similar. However, erythrocytes of the adult frog show a poor penetration by phenylhydrazine as indicated by the low spectral index obtained by treatment of eryt,hrocytes with 0.175 and 0.35 mM phenylhydrazine, i.e., 4.0 h 0.3 and 4.5 * 0.2 respectively. This observation is consistent with data reported later on 3-O-methylglucose transport by amphibian erythrocytes, in which the erythrocytes of tadpoles throughout all metamorphic stages and froglets transported this nonmetabolizable sugar quite
deviation. of 0.3 nmole
T, per gram
body
weight.
efficiently while the adult frog erythrocytes appear to have lost this property. The interaction of phenylhydrazine with each hemolysate was greater with prometamorphic tadpoles and T,-treated animals than for the other groups reported here as indicated by a higher spectral index. The injection experiments were designed to study the correlation between the in vivo hemolytic effect and in vitro spectral changes produced by phenylhydrazine in amphibians, i.e., speed and severity of the hemolytic phase with
414
DEVELOPMENTAL
BIOLOGY
availability of the drug in the target red cell. With the highest dose of phenylhydrazine used, 50 pg (0.35 pmoles/gm body weight), the largest spectral change was observed in froglets, correlating with the fastest hemolytic effect produced by phenylhydrazine in these animals (Fig. 1 and Table 1). Both the T,-treated tadpoles and the adult frogs showed a small spectral change, also in agreement with the relatively weak in uivo hemolytic effect of phenylhydrazine in these groups (Fig. 2 and Table 1, respectively.) Value of Hemoglobin
to Tadpole
27, 1972
and Frieden, 1959). When tadpoles were made anemic by immersion in 35 PM phenylhydrazine at room temperature, they survived at 30°C comparable to nonanemic controls. However, when tadpoles were immersed in 2,4-dinitrophenol, greater mortality was observed after they were pretreated with 35 PM phenylhydrazine. This effect was noted at both 21°C and 30 “C (Fig. 4), but, as expected, at a lower dinitrophenol concentration at 30 “C. It should be recognized that the use of mortality as a criterion does not distinguish between a response to the greater need for oxygen or to reinforcement of the toxicity caused by the use of both drugs. Nevertheless, the data may be interpreted as indicating that in the tadpole stressed with 2,4-dinitrophenol, Hb and red cells do contribute survival value to the tadpole.
Survival
Since the first observation of the longevity of completely anemic tadpoles (Flores and Frieden, 1968), the question of their survival in relation to Hb was raised. Since Grass0 and Shephard (1968) reported that newts treated with acetylphenylhydrazine showed an oxygen consumption 64-67s of controls, we attempted to devise experiments which led to an increase in the oxygen requirement of the tadpole. Earlier we reported that tadpoles have a higher oxygen utilization at 30” and after immersion in or injection of 2,4-dinitrophenol (Lewis 0
VOLUME
Postmetamorphic Effects on Facilitated Diffusion in Anumn Erythrocytes A brief study was made of the effects of development on the facilitated diffusion of 3-O-methylglucose into the anuran erythrocyte. The rate of uptake
No
+NHNH,
n With
35/.1M
@NHNH2
T= 21”
10
15
20
T=30°
L
25
2,4-DINITROPHENOL
30
10
15
(MM)
FIG. 4. Groups of 10 Ram catesbeiana tadpoles, stages X-XV, were made anemic by immersion in pM phenylhydrazine for 48 hr at 21 + 1”. They were then immersed in the indicated concentrations of 2,4-dinitrophenol at 21°C and 3O”C, respectively, and mortality was followed over a g-day period. Data obtained by Somjai Sirivech, Department of Chemistry, Florida State University.
FLORES
AND FRIEDEN
Hemolysis
of radioactive 3-0-methylglucose was found to be similar using erythrocytes obtained from the following animals: (1) normal human, (2) R. grylio and R. catesbeianu tadpoles, with or without incubation in 5 PM T, for 4 hr, (3) R. catesbeiana tadpoles after injection of 0.3 nmole T, per gram for l-11 days, and (4) R. catesbeianu froglets. In contrast, the erythrocytes of three adult anuran species. R. catesbeiana, R. pipiens, and toads and turtle erythrocytes showed less than 5% of the rate of uptake of the other 4 groups reported above. These data indicate that the changes in the permeability of 3-O-methylglucose is a postmetamorphic response with no change during or soon after metamorphosis. In this regard, the tadpole and froglet cells behave as do fetal erythrocytes from certain other mammalian species, e.g., pigs, rabbits, guinea pigs, comparable to adult human sheep, erythrocytes. These mammals lose the capacity for sugar transport in their erythrocytes after birth (Widdas, 1955). DISCUSSION
Induction of Anemia in Amphibiu by Phenylhydmzine The results presented in this paper indicate the differential sensitivity of amphibians to the hemolytic action of phenylhydrazine and the facility of these species in developing severe anemia without serious impairment to their survival. The percent survival, which may be related to the oxygen demands at any particular stage in metamorphosis and in adulthood, showed the following decreasing order: froglets > prometamorphic tadpoles > adult frogs > metamorphic climax tad:poles > T,-treated tadpoles. The oxygen needs in T,-treated and metamorphic climax tadpoles are higher than in prometamorphic tadpoles, which is in agreement with the higher mortality
and Anuran
Metamorphosis
415
in the first two groups as compared with the last one. Furthermore, the severe anemia induced by phenylhydrazine in prometamorphic tadpoles make these animals more susceptible to the lethal effect of 2,4-dinitrophenol than the corresponding controls. The survival of highly anemic newts has been explained mainly on the basis of increased pulmonary oxygen intake (Grass0 and Shephard, 19681, while in hemoglobin-less R. pipiens frogs (Meints, 1969) and icefish (Hemmingsen and Douglas, 1970) the compensatory mechanism seems to be an increase in blood volume and higher cutaneous respiration. The higher mortality observed when the animals were exposed to 2,4-dinitrophenol, T,-treatment and metamorphic climax may be explained in terms of limiting oxygen availability (Grass0 and Shephard, 1968). Differential Sensitivity of Anuran Erythroc) tes to Phenylhydrazine Many factors may contribute to the differential sensitivity of amphibians to the hemolytic action of phenylhydrazine including the possible role of the skin and gills in the absorption of the drug administered by immersion to the metamorphosing tadpoles (Fig. 1). The liver, which shows changes in numerous enzyme activities (see review by Frieden and Just, 1970) as well as qualitative and quantitative changes in its subcellular particles (Bennett et al., 1970a, b) during metamorphosis or after treatment of the tadpole with thyroid hormones must also play an important role in the detoxication of the drug. The interaction of phenylhydrazine with the amphibian erythrocyte may be affected by the differences in permeability of the red cell membrane at different stages of metamophosis and different rates of reaction with Hb once the drug has penetrated. Thus both membrane and cytoplasmic effects must be con-
416
DEVELOPMENTAL
BIOLOGY
sidered. The low spectral index obtained when adult frog erythrocytes were incubated with the drug (Table 2) is in agreement with the lower hemolytic effect of phenylhydrazine in the adult frog (Table 1). The shift in Hb during metamorphosis would not explain, by itself, the different sensitivity of amphibians to phenylhydrazine, since froglets showing the adult frog Hb patterns were the most sensitive to the drug (Table 1). Two alternative explanations may be offered to account for these results. It has been observed that 3-O-methylglucose is efficiently transported into the red cells of both tadpoles and froglets, while this ability is lost in the erythrocytes of the adult frog. This could be also true for phenylhydrazine, thus accounting for the low spectral index when frog erythrocytes were incubated with the drug (Table 2). The other alternative would be that, although the Hb is the same for the froglets and the adult frog, other components of these erythrocytes may differ, e.g., organic phosphates, reduced glutathione. Organic phosphates play an important role in the interaction of Hb with oxygen (Benesch et al., 1968; Salhany et al., 1970). OxyHb participates in the oxidation of phenyldiimide (an intermediate in phenylhydrazine metabolism), leading to the formation of peroxyHb, which in the presence of glutathione gives rise to the formation of Fe(III)-Hb derivatives with subsequent production of free radicals, which may be harmful to the red cell (Kosower et al., 1965). The fact that the rates of reaction of phenylhydrazine (followed at 410 nm and 630 nm) with lysates of tadpole and froglet erythrocytes differ considerably from that of the frog erythrocyte lysates supports this idea. Erythropoiesis
in Amphibia
Maniatis and Ingram (1971a) have recently shown that the liver is the organ where the erythroid cell matures both
VOLUME
27. 1972
before and during metamorphosis of R. catesbeianu tadpoles. The corresponding site in the frog is presumed to be the bone marrow. The rate of regeneration of Hb observed in this work was as follows: climax tadpoles > T,-treated tadpoles > adult frog > froglet > prometamorphic tadpoles is consistent with the delay in the onset of erythropoiesis (13-32 days) after acetylphenylhydrazine reported by Grass0 and Shephard (1968) in anemic newts. However, in the adult frog (Rana pipiens), erythropoiesis starts soon after the induced hemolytic phase (Meints, 1969). Although phenylhydrazine has toxic side effects in kidney and liver (Schuckmann, 1969), it may not damage other erythropoietic for the higher organs, thus accounting regenerative rate observed in froglets and adult frogs. If there is a shift in erythropoietic organs with active replacement of tadpole erythrocytes by the new frog erythrocyte line, the highest rate of regeneration would be expected for the tadpole at its metamorphic climax, which is consistent with our findings. The faster regeneration observed in the T,-treated tadpoles can be accounted for either by the loss of water from the intravascular compartment or true erythropoietic stimulation by thyroid hormones (Gallagher and Ford, 1969). It is interesting to note that the more time allowed between phenylhydrazine and T, administration, the higher the Hb concentration in those animals receiving hormonal treatment. This observation also supports the idea that reversible damage at the site of erythropoiesis plays a major role in determining the rate of regeneration. Effect of Phenylhydnxine globin Patterns
on Hemo-
The results reported in this paper indicate that the shift in Hb patterns which occur during metamorphosis is dependent only on the developmental
FLORES
.~ND FRIEDEN
Hem013 Isis and Anumn
stage of metamorphosing tadpoles. Contrary to what has been demonstrated mainly for some mammalian species, the severe anemia induced by phenylhydrazine did not produce any shift or preferential appearance of either fetal type (tadpole Hb) or abnormal Hb in the experimental animals studied in this work. However, phenylhydrazine treatment proved to be useful in showing the appearance of adult Hb patterns earlier than in appropriate controls. The appearance of adult Hb patterns (using phenylhydrazine) as early as stage XVIII in those tadpoles treated with a low dose of T, may be explained in terms of stimulation of spleen erythropoiesis as a consequence of anemia. It has been shown that in mammals, hypoxia (CO) stimulates spleen erythropoiesis (Ramsey, 1969). Since the bone starts developing at stage XX (Kemp and Hoyt, 1969), the earlier appearance of adult Hb at stage XVIII would also suggest the role of the spleen in producing frog Hb at this early stage in metamorphosis. This research was supported by U. S. Public Health Service Grant HD 01236 from the National Institutes of Child Health and Human Development. This is paper No. 44 in a series from this laboratory on the biochemistry of amphibian metamorphosis. REFERENCES BANERJEE, V., and BANERJEE, M. (1969). Seasonal variations of erythrocyte number and hemoglobin content in a common Indian lizard Vunanus monitor. 2001 Anz. 182, 203-207. BEAVEN, G. H., and WHITE, J. C. (1954). Oxydation of phenylhydrazine in the presence of oxyhemoglobin and the origin of Heinz bodies in erythrocytes. Nature (London) 173, 389-391. BENBASSAT, J. (1970). Erythroid cell development during natural amphibian metamorphosis. Deuelop. Biol. 21, 557-583. BENESCH, R., BENESCH, R. E., and Yu, C. I. (1968). Reciprocal binding of oxygen and diphosphoglycerate by human hemoglobin. Proc. Nat. Acad. Sci. U.S. 59, 526-532. BENNETT, T. P., GLENN, J. S., and SHELDON, H. (1970a). Changes in the fine structure of tadpole (R. catesbeianu) liver during thyroxine-induced metamorphosis. Deuelop. Biol. 22, 232-248.
Metamorphosis
417
BENNETT, T. P., and GLENN, J. S. (197Ob). Fine structural changes in liver cells of R. catesbeiana during natural metamorphosis. Deuelop. Biol. 22, 535-560. CAMERON, J. N., and WOHLSCHLAG, D. E. (1969). Respiratory response to experimentally induced anemia in the pinfish (Lagodon rhomboides). J. Exp. Biol. 50, 307-317. DEGRAAF, A. R. (1957). A note on the oxygen requirements of Xenopus laeuis. J. Exp. Biol. 34, 173-176. DEWITT, W. (1968). Microcytic response to thyroxine administration. J. Mol. Viol. 32, 502-504. EWER, D. W. (1959). A toad (Xenopus laeuis) without haemoglobin. Nature (London) 183, 271. FLORES, G. (1969). Hemolytic and spectral effects of phenylhydrazine and hydrazine derivatives in amphibian red cells. M. S. Thesis. The Florida State University, Tallahassee, Florida. FLORES, G., and FRIEDEN, E. (1968). Induction .and survival of hemoglobin-less and erythrocyte-less tadpoles and young bullfrogs. Science 151, 101-103. FLORES, G., and FRIEDEN, E. (1970). Structural requirements for the hemolytic effect of phenylhydrazine derivatives on amphibian red cells. J. Pharmacol. Exp. Ther. 174, 463-472. FRIEDEN, E., and JUST, J. J. (1970). Hormonal responses in Amphibian metamorphosis. In “Biochemical Actions of Hormones” (E. G. Litwack, ed), pp. l-52. Academic Press, New York. FRIEDMAN, G. B., ALGARD, F. T., and MCCURDY H. M. (1969). Determination of red blood cell count and hemoglobin content of urodele blood. Anut. Rec.163, 55-57. GALLAGHER, N. I., and FORD, G. H. (1969). Effect of hyperoxia on the erythropoietic response of sodium L-triiodothyronine. Proc. Sot. Exp. Biol. Med. 130, 672-674. FOLDSTEIN, G. W., HAMANAKER, L., and SCHMID, R. (1968). The catabolism of Heinz bodies: An experimental model demonstrating conversion to non-bilinium catabolites. Blood 31, 388-395. GRASSO, J. A., and SHEPHARD, D. C. (1968). Experimental production of totally anemic newts. Nature (London) 218, 1274-1276. HEMMINGSEN, E. A., and DOUGLAS, E. L. (1970). Respiratory characteristics of the hemoglobinfree fish (Chaemocephalus acemtus). Comp. Biochem. Physiol. 33, 733-744. HERNER, A. E., and FRIEDEN, E. (1961). Biochemical changes during anuran metamorphosis. VIII. Changes in the nature of red cell proteins. Arch. Biochem. Biophys. 95, 25-35. HOLLYFIELD, J. G. (1966). Erythrocyte replacement at metamorphosis in the frog, R. pipiens. J. Morphol. 119, 1-6. JANDL, J. H. (1963). The Heinz body hemolytic anemias. Ann. Intern. Med. 58, 702-709.
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DEVELOPMENTAL
BIOLOGY
KEMP, N. E., and HOYT, J. A. (1969). Sequence of ossification in the skeleton of growing and metamorphosing tadpoles of Rana pipiens. J. Morphol. 129, 415-443. KOSOWER, N. S., VANDERHOFF, G. A., KOSOWER, E. M., and HUANG, P. C. (1965). Decreased glutathione content of human erythrocytes produced by methyl phenylazoformate. Biochem. Biophys. Res. Commun. 20, 469-474. LEMBERG, R., and LEGGE, J. W. (1949). “Hematin Compounds and Bile Pigments: Their Constitution, Metabolism, and Function,” pp. 524-528. Wiley (Interscience) New York. LEWIS, E. J., and FRIEDEN, E. (1959). Biochemistry of amphibian metamorphosis: Effect of triiodothyronine, thyroxin, and dinitrophenol on the respiration of the tadpole. Endocn’nology 65, 273-282. MANIATIS, G. M., and INGRAM, V. M. (1971a). Erythropoiesis during amphibian metamorphosis. I. Site of maturation of erythrocytes in Rano catesbeiam. J. Cell Biol. 49, 372-379. MANIATIS, G. M., and INGRAM, V. M. (1971b). Erythropoiesis during amphibian metamorphosis. II. Immunochemical study of larval and adult hemoglobins of Rana catesbeianu. J. Cell Biol. 49, 380-389. MANIATIS, G. M., and INGRAM, V. M. (1971c). Erythropoiesis during amphibian metamorphosis. III. Immunochemical detection of tadpole and frog hemoglobins (Rana catesbeiuna) in single erythrocytes. J. Cell Biol. 49, 390-404. MANIATIS, G. M., STEINER, L. S., and INGRAM, V. M. (1969). Tadpole antibodies versus frog hemoglobin and their effect on development. Science 165, 67-69. MEINTS, R. (1969). Erythropoietic activity in Ranu pipiens. The influence of severe hemolytic induced anemia on spleen and peripheral blood activities. Comp. Biochem. Physiol. 30, 383-389. MILLS, G. C., and RANDALL, H. P. (1958). Hemo-
VOLUME
27. I972
globin catabolism. II. The protection of hemoglobin from oxidative breakdown in the intact erythrocyte. J. Biol. Chem. 232, 589-598. Moss, B., and INGRAM, V. M. (1968a). Hemoglobin synthesis during amphibian metamorphosis. I. Chemical studies on the hemoglobins from the larval and adult stages of Ranu catesbeiana J. Mol. Biol. 32, 481-492. Moss, B., and INGRAM, V. M. (1968). Hemoglobin synthesis during amphibian metamorphosis. II. Synthesis of adult hemoglobin following thyroxine administration. J. Mol. Biol. 32, 493-504. RAMSEY, J. M. (1969). Immediate hematological response to the rat to exposure to CO. J. Physiol. (London) 202, 297-304. RUDD, J. R. (1954). Vertebrates without erythrocytes and blood pigments. Nature (London) 173, 848-850. RUGH, R. (1962). “Experimental Embryology” p. 50. Burgess, Minneapolis, Minnesota. SALHANY, J. M. ELIOT, R. S., and MIZUKAMI, H. (1970). Effect of 2,3-diphosphoglycerate on the kinetics of deoxygenation of human hemoglobin. Biochem. Biophys. Res. Commun. 39, 1052. SCHUCKMANN, F. (1969). Different forms of phenylhydrazine intoxication. Zentrolbl. Argeitsmed. Arbeitsschutz 19, 338-341. SULLIVAN, B., and RIGGS, A. (1964). Haemoglobin: Reversal of oxidation and polymerization in turtle red cells Nature (London) 204, 1098-1099. TAYLOR, A., and KOLLROS, J. J. (1946). Stages in normal development of Ranu pipiens larvae. Anat. Rec. 94, 7-23. THEIL, E. C. (1967). The synthesis of hemoglobin and non-hemoglobin protein of amphibian red blood cells related to metamorphosis. Biochim. Biophys. Acta 138, 175-185. WIDDAS, W. F. (1955). Hexose permeability of foetal erythrocytes. J. Physiol. (London) 127, 318-327.