The effects of variously substituted quinoxalines, benzimidazoles, and benzotriazoles on Rana pipiens embryos of 2-cell (S. 3), blastula (S. 8), neurula (S. 14) and tail-bud stage (S. 18) were studied. It was found that:

  1. Toxicity is variably increased by double substitution with two nitro groups (diN) or a nitro group and a chlorine atom (N+Cl). The relative toxicity of the single N-substituent is diminished by the addition of a hydroxy group, and generally also by a methoxy group, but not by addition of a methyl group; still greater is the diminishing effect of the carboxyl group which nullifies all activity. Cl-substitution in place of N is less toxic except at stages 3 and 8 with benzimidazoles.

  2. Susceptibility to benzo triazoles increases with age of embryo from 2-cell to tail-bud stage 18, while the reverse is true for quinoxalines and benzimidazoles. The diN-or N+Cl-substituted compounds are exceptions, as they are highly toxic at all developmental stages under the experimental conditions.

  3. In young tadpoles (S. 25) N-substitution increases the toxicity of quinoxaline or benzimidazole approximately 10 times, and of benzotriazole as much as 40 times.

Substituted benzotriazoles (Bt) have been shown to bring about interesting inhibitory effects on Rana pipiens embryos of 2-cell to tail-bud stages (Liedke, Engelman, & Graff, 1954, 1955, 1957a, 1957b). These benzotriazoles did not have selective cytotoxic effects on sensitive embryonic structures as similarly substituted benzimidazoles (Bz) and quinoxalines (Q) invariably did. The latter compounds, Bz and Q, were most active against younger stages, especially those in cleavage. On the other hand, it was found that the susceptibility to the benzotriazoles increased with age of embryo ; more differentiated stages were affected most. The type of response was determined by the parent structure, but certain substituents, the nitro group in particular, appeared to enhance the magnitude of the effect. The activating effect of the nitro group was in turn modified to varying degree by an accompanying methoxy, hydroxy, or amino group.

It appeared desirable to study these modifying effects further by employing derivatives of the three classes of compounds, in which (a) a chlorine atom replaced the nitro group, and (b) in which the nitro group is accompanied by either a chlorine atom, a methyl, a carboxyl, or a second nitro group. Embryos at the 2-cell, blastula, neurula, or tail-bud stages and tadpoles (stages 3, 8, 14, 18, and 25, according to Shumway, 1940) were exposed to the test compounds. In addition to the Bz, Bt, and Q compounds, four other compounds (2,3-diamino-5-nitro-benzoic acid, 3,4-diamino-5-nitro-benzoic acid, 3,4-diamino-2-nitro-anisole, and 5-chloro-3-nitro-o-phenylene diamine) were tested similarly.

The methods were the same as those described in an earlier communication (Liedke, Engelman, & Graff, 1955). For a given experimental series, embryos from the same egg-batch were used. Exposure to the compounds (10 embryos per 20 ml. of test solution per stender dish) was started when the embryos had developed to stages 3, 8, 14, and 18. All solutions were made up in 10 per cent. Ringer solution and maintained at approximately pH 6·7 (phosphate buffer). At the concentration used (50 μg./ml.) the compounds have little or no effect on pH. Temperature varied from 20 to 22° C. in the experiments using continuous exposure, and from 22 to 24° C. in the experiments where the length of exposure was varied. Instances of low solubility of test compounds are noted in the table.

Egg-batches from 10 different females were used in the experimental series. Different egg-batches varied somewhat in their response to the compounds, but the results were consistent when all the compounds were tested on the same batch. Varying lengths of exposure were employed with 6 batches of eggs also, in conformity with our previously reported finding that the severity of effect depends on the length of exposure as well as on concentration.

In Table 1 are fisted a total of 34 compounds employed in the experiments at a concentration of 50 μg./ml. (except where solubility was limited) in the experiments on young embryos, and at a considerably lower concentration in the experiments on tadpoles (see below). Six of these compounds had been used in earlier experiments (Liedke, Engelman, & Graff, 1954, 1951b) and were used again to broaden the base of comparison.

TABLE 1.

Stage of arrest in development of Rana pipiens embryos after continuous exposure to 50 μg./ml. of various substituted benzimidazoles (Bz) and benzotriazoles (Bt)

Stage of arrest in development of Rana pipiens embryos after continuous exposure to 50 μg./ml. of various substituted benzimidazoles (Bz) and benzotriazoles (Bt)
Stage of arrest in development of Rana pipiens embryos after continuous exposure to 50 μg./ml. of various substituted benzimidazoles (Bz) and benzotriazoles (Bt)

Experiments on embryos in 2-cell, blastula, neurula, and tail-bud stages

Continuous exposure

The data in Table 1 show that:

  • (1) Toxicity was markedly enhanced where either the benzotriazole or benzimidazole nucleus was nitro-substituted, as had been found before. Unsubstituted quinoxaline is highly toxic in itself, and nitro substitution affected it but little.

  • (2) Chlorine substitution enhanced toxicity of the parent compound much less than did nitro substitution, except at S. 3 with 5-Cl-Bz, which was highly toxic.

  • (3) Double substitution with nitro and chloric groups or with two nitro groups produced variable toxicity depending on the parent compound.

  • (4) The introduction of a methyl group to a nitro-substituted compound usually decreased the toxicity to older stages, but not at all when stage 2 embryos were treated.

  • (5) The addition of a carboxyl group to a number of compounds was most decisive, completely abolishing toxicity in most instances.

Not listed in Table 1 are four compounds which were also tested. Three of these had no effect on the embryos, namely: 2,3-diamino-5-nitro-benzoic acid, 3,4-diamino-5-nitro-benzoic acid, and 3,4-diamino-nitro-anisole; but continuous exposure to 50 μg./ml. of 5-chloro-3-mtro-o-phenylene diamine arrested 2-cell stages at S. 12–15, blastulae at S. 15, neurulae at S. 18–19, and tail-buds at S. 20.

Varied length of exposure

Varied length of exposure (Table 2) using the same concentration (50 μg./ml.) at 22–24° C. generally confirmed the results given in Table 1, but it also permitted some finer distinction.

TABLE 2.

Stage of arrest in development of Rana pipiens embryos after 1, 2, 8, or 24 hours of exposure to 50 μg. /ml. of various substituted benzotriazoles (Bt), benzimidazoles (Bz), and quinoxalines (Q)

Stage of arrest in development of Rana pipiens embryos after 1, 2, 8, or 24 hours of exposure to 50 μg. /ml. of various substituted benzotriazoles (Bt), benzimidazoles (Bz), and quinoxalines (Q)
Stage of arrest in development of Rana pipiens embryos after 1, 2, 8, or 24 hours of exposure to 50 μg. /ml. of various substituted benzotriazoles (Bt), benzimidazoles (Bz), and quinoxalines (Q)

Table 2 shows that Cl-N-Bt is the most toxic compound, except at the 2-cell stage (S. 3) where 1 hour of exposure permits development to stage 11, while N-or Cl-N-Q-treated 2-cell stages are stopped already at S. 3–5. But longer exposure to Cl-N-Bt (2-8 hours) also stops development earlier, at S. 5–6.

Table 2 also shows that N-or Cl-N-substituted quinoxaline is more toxic than Q (i.e. it requires only 1 hour of exposure, while Q requires 2-8 hours to arrest at stages 4–5). On exposure at later stages (S. 8, 18) this difference is even more apparent, and it is also seen that Cl-N-Q is more toxic than N-Q. It is seen, furthermore, that at later stages, S. 8, and even more at S. 18, longer exposures are required for Q-compounds than at S. 3, while almost the reverse is true for substituted Bt-compounds. However, it should be noted that Bt-compounds (except the double substituted diN-or Cl-N ones) require longer exposures than Q-or most Bz-compounds at all stages, i.e. at least 8 hours of treatment are required. Nevertheless, allowing 8–24 hours of treatment, even the single substituted benzotriazoles acted more severely at later than at earlier developmental stages.

The diN-substituent is generally more toxic than single N-in Bt and Bz (cf. columns 8 and 24 hours in Table 2). Cl-in place of N-is definitely less toxic in Bt, but that is reversed in Bz, and the order of toxicity is Cl-Bz > N-Bz, particularly at stages 3 and 8. This difference is more apparent in Table 2 than in Table 1. Nevertheless, paradoxically, the added Cl-or double substitution (Cl-N-Bz) is less toxic than either of the single ones (N-Bz or Cl-Bz). On the other hand, double Cl-N in Bt is extremely toxic, and Cl-N-Bt is the most toxic compound of all the ones used, as will be shown below in experiments with 11-mm. tadpoles (Table 3). One could have expected the opposite, namely, that Cl-N-Bz rather than Cl-N-Bt would be the most toxic compound if it were a matter of simply adding ‘more toxic C1-’ to N-Bz, and Tess toxic C1-’ to N-Bt.

TABLE 3.

The effect of C1-NO2 or NO2-substituents in benzotriazole (Bt), benzimidazole (Bz), or quinoxaline (Q) on Rana pipiens embryos (stage 25)

The effect of C1-NO2 or NO2-substituents in benzotriazole (Bt), benzimidazole (Bz), or quinoxaline (Q) on Rana pipiens embryos (stage 25)
The effect of C1-NO2 or NO2-substituents in benzotriazole (Bt), benzimidazole (Bz), or quinoxaline (Q) on Rana pipiens embryos (stage 25)

Experiments with 10–11-mm. tadpoles (S. 25)

For each experimental series tadpoles from the same egg-batch were exposed to the three unsubstituted parent compounds and the N-substituted ones, and also to Cl-N-Bt (5 tadpoles per 20 ml. of test solution per large stender dish, or 10 per 40 ml. per finger-bowl).

The great toxicity of the substituted benzotriazoles on older embryos became rather troublesome in the work with these tadpoles (S. 25) which are 6 days older than the tail-bud embryos (S. 18) used in the above-described experiments (Tables 1, 2). Survival was irregular and unpredictable even at very low concentrations of N-Cl-Bt or N-Bt (Table 3). The tadpoles were normal and active after the brief exposure to N-Cl-Bt or N-Bt, when they were rinsed of the test solution and transferred to 10 per cent. Ringer solution. Sometimes it seemed that their motility was even greater or slightly spastic compared to the controls.

Nevertheless, motility often ceased within 2–6 hours after treatment, and very soon thereafter the heart stopped beating. On very rare occasions some individuals survived and continued normal growth like the controls. Neither the volume of blood in the heart nor gill circulation can be observed in these tadpoles because of overgrowth of the operculum. However, in simultaneous identical experiments with younger tadpoles (S. 23) it was noticed that the gill circulation had become more sluggish and sometimes stopped during the first day after treatment.

Such sudden death, or only occasional survival of some individuals, was not found with N-Bz or N-Q. Rather the same concentration (20 μg./ml.) and even continuous exposure to N-Bz or N-Q permitted survival from 2 to 5 days, and for longer if exposure was for 5–6 hours (compared to 1–2 hours with N-Bt) as seen in Table 3. Only slight or no oedema developed in these tadpoles exposed to 20 μg./ml. N-Bz or N-Q for 5-6 hours. Three days later the eyes showed some loss of pigment, particularly in the N-Q-treated tadpoles, which were not studied histologically. It is therefore not possible to state how much necrosis and possible regeneration occurred. Loss of eye pigment can indicate regeneration if necrosis is stopped, and the tadpole is notoedematous. With continuous exposure to 20 μg./ml. N-Bz or N-Q the tadpoles became progressively oedematous and showed poor motility, and death occurred between 2 and 5 days after start of exposure. The same results were obtained with the three unsubstituted parent compounds when the concentration was increased tenfold, i.e. 200 μg./ml. of N-Bz, N-Bt, or N-Q. Tadpoles from these experiments (continuous exposure to 200 μg./ml. Bz, Bt, or Q, or to 20 μg./ml. of the N-substituted compounds) were studied histologically.

Histological observations

For histological study some tadpoles were killed 4 hours or 1, 2, or 3 days after initiation of the treatment. Harris haematoxylin, phosphatungstic acid haematoxylin, and eosin-azure stains were used after fixation in Bouin, Zenker, or Helly fluid. Embryos from some of the younger series were also prepared for histological study.

In previous work (Liedke, Engelman, & Graff, 1955, 1957b) the characteristic response of these younger embryos (stages 3, 8, 14, and 18) to Bt-compounds always involved a generalized effect, namely, sudden death, or delay in development leading to certain malformations depending on the developmental stage treated. A localized cytotoxic effect, like selective necrosis or enlargement of cells in sensitive embryonic structures, was not found in spite of abnormal development and some microcephaly. Such a localized cytotoxic response, on the other hand, always occurred with quinoxalines and benzimidazoles. In this study, however, a localized cytotoxic response was seen also in Cl-substituted benzotriazoles in these younger embryos (stages 3, 8, 14, and 18).

Histological study of 11-mm. tadpoles treated with all three compounds always showed localized selective cellular necrosis in the eye, central nervous system, head cartilage, blood-cells, and the liver. The N-substituted compounds were more effective than the unsubstituted ones; N-substituted benzotriazole was more toxic than N-substituted benzimidazole or quinoxaline, and N-Cl-Bt was extremely toxic (Table 3).

One would expect localized cytotoxic responses in 11-mm. tadpoles to any interfering agent. The principal organ systems are present, although growth and differentiation still continue, and some structures, limbs, spleen, gonads, &c., have not as yet developed. The circulatory system functions and is now of primary importance, even though intracellular utilization of yolk still provides metabolites. The number of blood-cells increases constantly. It is, therefore, not surprising that not only the sensitive eye and nervous system, as in younger developmental stages, should be strongly affected, but also the blood-cells and liver.

Two or three days after exposure the muscular activity of the tadpoles was poorer and oedema had developed. The heart continued beating at a normal rate but the volume of blood seemed to be less. Unfortunately, gill-circulation changes could not be observed because of overgrowth of the operculum; however, shrinkage of gills was apparent in sections of some tadpoles.

The most severe effect prior to death was complete failure of the circulatory system and advanced necrosis in the Ever. This is illustrated in Plate 1, fig. A, Plate 2, fig. F, which show transverse sections of a tadpole 3 days after continuous exposure to 0·020 μg./ml. of unsubstituted benzotriazole. When the circulation is disrupted, the lens becomes pressed into the eye-cup, while vascular spaces between the eye and brain, and capillaries in the brain, are obliterated, as a comparison between figs. A, B, C of Plate 1 shows. Not only is the number of blood-cells reduced, but necrotic blood-cells are everywhere evident, particularly in the liver. The liver-cells are also dying and the pronephric tubules have become distended (Plate 2, figs. E, F). At the same time, pycnotic nuclei are present in the cartilage of the head and the nerve-cord, but the most extensive necrosis and cell debris are found in the ventral portion of the brain. Most cells, particularly those in the eye, stain poorly (Plate 1, fig. A). All compounds elicited this severe cytotoxic response, but the effectiveness or toxicity varied greatly (N-Bt > NQ or N-Bz > Bt or Q), as seen in Table 3.

It was possible to trace the histological events which led to this severe cytotoxic response by fixing the tadpoles at different time intervals (4–72 hours after initiation of treatment with N-Bz, N-Bt, N-Q, Bt, or Q). The earliest abnormalities were noticed in the eye, usually on the first day after treatment, as seen in Plate 1, fig. C, which is a transverse section through the eye of a tadpole 1 day after exposure to 20 μg./ml. N-Q. A few pycnotic nuclei (n) are seen here, lying next to normal mitoses (m) in the iris angle, the zone of most rapid proliferation and earliest cellular differentiation. While normal tadpoles also have a few degenerating nuclei in the eye and central nervous system, such pycnotic nuclei occurred more frequently in N-Q eyes, and earlier than blood-cell destruction. In this particular specimen, however, localized necrosis of a few blood-cells (nB) was seen in a few sections through the choroid plexus (Plate 2, fig. H). The pigment cells (P) are also affected and contracted. This effect on pigment cells occurred frequently with N-Q, even when there was no localized disturbance in the blood-cell population, but not with N-Bz nor Bt.

A more severe effect is usually found on the second or third day, as seen in Plate 1, fig. B, a transverse section through the eye region of a tadpole 2 days after exposure to 20 μg./ml. N-Bz. The number of necrotic cells (n) in the iris angle has now increased, mitotic figures are absent, and pycnotic nuclei are also scattered in the brain, adjacent to the ependymal layer. There is no visible involvement of blood-cells, in fact, the capillaries of brain and eye contain normal blood-cells, as Plate 1, fig. B shows. However, a few groups of degenerating blood-cells (nB) are scattered among the normal ones in the liver sinuses of this tadpole (Plate 2, fig. G).

Necrotic areas in the brain increased with time in size and number, extending to posterior levels, and pycnotic nuclei appeared in the head cartilage; the number of necrotic blood-cells increased in the liver sinuses and began also to appear in the pronephric sinuses. All gradations from slight to severe destruction of sensitive cells in the eye, the central nervous system, and cartilage, as well as of blood-cells, were found in tadpoles treated continuously with N-Q, N-Bz, or Bt, and this led finally to liver necrosis (Plate 1, fig. A; Plate 2, fig. F) as described above.

With shorter exposure (5–6 hours in 20 μg./ml. N-Q or N-BZ) only slight or no oedema developed; survival time was increased (Table 3). However, the results were always unpredictable with N-Bt, as described above. Histological study showed that some of the surviving N-Bt-treated tadpoles were normal; in other survivors of normal appearance necrotic cells in the eye or brain were still present 24 hours later (Plate 1, fig. D). This tadpole, however, has a normal liver (Plate 2, fig. E) with many mitotic cells.

These experiments again illustrate the influence of type, number, and position of substituents on biological activity. The benzo triazoles, benzimidazoles, and quinoxalines shown in Text-fig. 1 are rather stable resonating structures. Their biological reactivity probably involves nitrogen atoms 1 and 3 in Bz and Bt, and 1 and 4 in Q.

TEXT-FIG. 1.

Structural formulae of Bz, Bt, and Q, a representative purine, and riboflavin. (Chemical Abstracts numbering system.)

TEXT-FIG. 1.

Structural formulae of Bz, Bt, and Q, a representative purine, and riboflavin. (Chemical Abstracts numbering system.)

The Bz and Bt compounds could theoretically compete with purines for nucleoside formation by virtue of the similarity between nitrogen atom 1 and the pyridine nitrogen atom in purine position 9, and, indeed, Woolley (1944) and Kidder et al. (1949) have provided support for such an hypothesis by reversing the toxicity of several substituted benzimidazoles with purines in bacteria and protozoa. However, reversal has not been observed in metazoa, including frog embryos (Manikis, 1959) and chick embryos (Billet & Perry, 1957a, b).

It is usual for the strongly negative nitro or chloro loadings to enhance toxicity in compounds of this type. This was also observed in Arbacia eggs where chloro substitution in caffein was more inhibitory than ethoxy or methyl substitution (Cheney, 1957). The introduction of a carboxyl group into heterocyclic nitrogen compounds almost invariably diminishes toxicity, as it did decisively in our experiments, except for 2,3-diPh-7-N-5-Cx-quinoxaline.

The quinoxalines, on the other hand, can be likened to riboflavin, and conceivably compete with it for the formation of flavin nucleotides, which are presumably essential for development. We have not, however, carried out reversal experiments to test this possibility. It would not be anticipated that substitution in the benzene moiety would have much effect on the reactivity of quinoxaline while 2,3-substitution should decrease reactivity. It does so in our experiments at stage 3, but not at the older stages (Table 1). However, the reverse is true for 2,3-diMe-6-MO-5-N-Q, which is toxic for the embryos, while 6-MO-5-N-Q permits normal development. This latter compound illustrates also the effect of position; 6-MO-5-N-Q is non-toxic, in contrast to 7-MO-5-N-Q and 5-MO-7-N-Q, both of which are toxic, as previously reported (Liedke, Engelman, & Graff, 1957a, b).

Assessment of the relation of structure to biological activity is complicated further by unanswered questions on solubility and transfer to the interior of the treated structures wherein metabolic activities are compartmentalized. The purines, riboflavins, folic acid, and vitamin B12 all play a role in development, but their concentrations, cellular sites, and rates of use are unknown and cannot be related to specific morphological events.

It has been observed in disrupted staphylococcus cells, on the other hand, that derivatives of benzimidazole in which the 5,6 positions are substituted by CH3, Cl, or NO are potent inhibitors of glycine incorporation, but that 6-amino-4-hydroxy benzimidazole is an activator, suggesting that the actions of the inhibitory derivatives are due to structural analogies with benzimidazoles rather than with purines (E. F. Gale, personal communication).

Réactions de Vembryon à des inhibiteurs ayant une parenté structurale II Les effets des substituants

Les effets de substituants variés des quinoxalines, des benzimidazoles, des benzotriazoles ont été étudiés sur les embryons de Rana pipiens aux stades 2 blastomères (St. 3), blastula (St. 8), neurula (St. 14) et bourgeon caudal (St. 18). Les résultats ont été les suivants :

  1. La toxicité s’accroît d’une manière variable par la double substitution avec 2 groupes nitro (di-N) ou un groupe nitro et un atóme de chlore (N+Cl). La toxicité relative du simple substituant N est diminué par l’addition d’un groupe hydroxyl, et généralement aussi par un groupe méthoxyl; la diminution de l’effet toxique est encore plus importante avec le groupe carboxyl, qui annulle toute activité. La substitution de Cl à l’emplacement d’un N est moins toxique, sauf aux stades 3 et 8, dans le cas des benzimidazoles.

  2. La sensibilité aux benzotriazoles s’accroît avec l’âge de l’embryon du stade 2 blastomères au stade du bourgeon caudal (St. 18), tandis que l’inverse est vrai pour les quinoxalines et les benzimidazoles. Les composés substitués par un di-N ou par N-j-Cl sont des exceptions, car ils sont extrêmement toxiques à tous les stades du développement dans les conditions expérimentales.

  3. Chez les jeunes têtards (St. 25), la substitution de N augmente la toxicité des quinoxalines et des benzimidazoles d’environ 10 fois, celle des benzotriazoles d’au moins 40 fois.

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Plate 1

FIGS. A-D. Photographs of transverse sections through the eye and forebrain region of 11-mm. experimental tadpoles. Magnifications × 100, except for fig. C (×400).

FIG. A. After 3 days in 20 μg./ml. benzotriazole (Bt). Note massive necrosis in ventral portion of brain, isolated pycnotic nuclei (n) in retina and cartilage, and simultaneous circulation failure, i.e. blood-vessels obliterated and lens pressed into eye-cup. Liver necrosis seen in fig. F.

FIG. B. After 2 days in 20 μg./ml. N-Bz. Note necrosis of proliferating and early differentiating cells in iris angle, pycnotic nuclei in brain, no disturbance in circulation except for some necrotic cells in liver sinuses shown in fig. G.

FIG. C. After 1 day in 20 μg./ml. N-Q. Note only a few pycnotic cells (n) in iris angle; normal circulation and liver, except for choroid plexus shown in fig. H.

FIG. D. After 1 day, following 70 minutes exposure to 20 μg./ml. N-Bt. Note that absorption of necrotic cells in the thinner retina and the brain is still incomplete; normal blood-cells and liver (fig. E).

Plate 1

FIGS. A-D. Photographs of transverse sections through the eye and forebrain region of 11-mm. experimental tadpoles. Magnifications × 100, except for fig. C (×400).

FIG. A. After 3 days in 20 μg./ml. benzotriazole (Bt). Note massive necrosis in ventral portion of brain, isolated pycnotic nuclei (n) in retina and cartilage, and simultaneous circulation failure, i.e. blood-vessels obliterated and lens pressed into eye-cup. Liver necrosis seen in fig. F.

FIG. B. After 2 days in 20 μg./ml. N-Bz. Note necrosis of proliferating and early differentiating cells in iris angle, pycnotic nuclei in brain, no disturbance in circulation except for some necrotic cells in liver sinuses shown in fig. G.

FIG. C. After 1 day in 20 μg./ml. N-Q. Note only a few pycnotic cells (n) in iris angle; normal circulation and liver, except for choroid plexus shown in fig. H.

FIG. D. After 1 day, following 70 minutes exposure to 20 μg./ml. N-Bt. Note that absorption of necrotic cells in the thinner retina and the brain is still incomplete; normal blood-cells and liver (fig. E).

Plate 2

FIGS. E–H. Photographs of transverse sections through the liver or forebrain of the same experimental tadpoles as shown in figs. A–D. Magnifications x 300, except for fig. F (× 100).

FIG. E. After 1 day, following 70 minutes exposure to 20 μg./ml. N-Bt. Note active mitoses of blood-cells in liver and normal pronephric tubules.

FIG. F. After 3 days in 20 μg./ml. Bt. Note complete circulation failure, liver necrosis, and distension of pronephric tubules.

FIG. G. After 2 days in 20 μg./ml. N-Bz. Note pycnotic and normal blood-cells in liver sinuses.

FIG. H. After 1 day in 20 μg./ml. N-Q. Note necrosis of blood-cells (nB) in choroid plexus and contraction of pigment cells (p).

Abbreviations: h = hypertrophied cell; n = necrotic cell ; nB = necrotic blood-cell; tn = mitotic cell; p = pigment cell; Pro = Pronephros.

Plate 2

FIGS. E–H. Photographs of transverse sections through the liver or forebrain of the same experimental tadpoles as shown in figs. A–D. Magnifications x 300, except for fig. F (× 100).

FIG. E. After 1 day, following 70 minutes exposure to 20 μg./ml. N-Bt. Note active mitoses of blood-cells in liver and normal pronephric tubules.

FIG. F. After 3 days in 20 μg./ml. Bt. Note complete circulation failure, liver necrosis, and distension of pronephric tubules.

FIG. G. After 2 days in 20 μg./ml. N-Bz. Note pycnotic and normal blood-cells in liver sinuses.

FIG. H. After 1 day in 20 μg./ml. N-Q. Note necrosis of blood-cells (nB) in choroid plexus and contraction of pigment cells (p).

Abbreviations: h = hypertrophied cell; n = necrotic cell ; nB = necrotic blood-cell; tn = mitotic cell; p = pigment cell; Pro = Pronephros.