Embryotoxicity
As a first step in the characterization of the toxicity of methylxanthine compounds, we studied the lethality and phenotypic effects.
Five increasing doses/graded doses of eight methylxanthines: aminophylline, caffeine, diprophylline, doxofylline, etophylline, 3-isobutyl-1-methylxanthine (IBMX), pentoxifylline and theophylline; were injected into the yolk of 1-2 cell zebrafish embryos (Table 1) and the survival and phenotype analyzed at specific timepoints. The doses were selected in such a way that they fall within per oral therapeutic range in human. As the weight of zebrafish embryos is roughly 1.2 mg, administering 1 ng of a drug in one embryo (1 ng/1.2 mg) is approximately equal to 1 mg/kg of drug. Therefore, the doses of drugs used in microinjection were in the range of 1-20 mg/kg and most of these drugs have per oral therapeutic dose that falls within this range in humans.
Aminophylline, caffeine, doxofylline, IBMX, pentoxifylline and theophylline treated embryos showed dose dependent survival at 72 hpf, whereas the survival of diprophylline and etophylline treated embryos was independent of the doses (Fig. 1). IBMX was the most toxic/active compound: a large percentage of the embryos (58%) showed mortality when injected with 1 ng of the drug. Diprophylline was the least active methylxanthine; with more than 90% of embryos surviving at dose as high as 20 ng/embryo. Pentoxifylline and caffeine also produced >50% mortality, although they required higher doses than IBMX, i.e. 3 ng and 2.5 ng per embryo respectively to produce the equivalent mortality.
To simplify the comparison, we observed the dose at which there was a minimum of 20% mortality for each drug. Methylxanthines like IBMX, caffeine, pentoxifylline and theophylline were highly active with <1 ng needed to induce 20% mortaility. Aminophylline, etophylline and doxofylline were moderately active with 2.5-5 ng required to induce 20% mortality and diprophylline was a non toxic compound which didn’t induce 20% mortality even with the highest dose of 20 ng. To further compare the toxicity of each individual compound, we calculated the lethal dose at which there was 50% mortality (LD50) for each drug using linear interpolation and extrapolation. Our results showed that IBMX was the most toxic drug (LD50 = 0.91 ng) followed by caffeine (2.4 ng), pentoxifylline (2.76 ng), theophylline (3.4 ng) and aminophylline (6.15 ng), etophylline (9.8 ng),doxofylline (29.3 ng) and diprophylline (167.5 ng).
General Teratogenicity and general morphological defects
After evaluating the mortality, the alive embryos were observed for morphological defects at 48 and 72 hpf. The embryos with morphological defects were identified and classified into mild and severe phenotype based on the presence of different morphological endpoints (Table 2). In particular, embryos with defective heart formation, irregular heartbeat, decreased blood circulation, mild pericardial and yolk sac edema were categorized as mild phenotype. Embryos with perturbed anterior-posterior axis development with reduced tail detachment and poor formation of somites, defective heart formation, irregular heartbeat, absent blood circulation, severe pericardial and yolk sac edema were categorized as severe phenotype.
The percentage of embryos that showed morphological defects with the highest concentration of drug varied with each compound. While the lowest dose used produced morphological defects in <10% of the embryos, the highest concentration produced morphological defects in 50% of embryos with 2.5 ng of caffeine, 44% with 1 ng of IBMX, 38% with 3 ng of pentoxifylline, 29% with 5 ng of aminophylline, 24% with 1 ng of theophylline, 23% with 7.5 ng of doxofylline, 15% with 5 ng of etophylline and 5% with 20 ng of diprophylline. The embryos injected with the positive control DCA showed 47% morphological defects, while less than 1% of negative controls injected with sterile water showed morphological defects (Fig. 2). These embryos predominantly had pericardial edema, abnormal blood circulation, yolk sac edema and abnormal AP axis formation (Fig. 3).
For the comparison of the morphological defects, we looked into the dose required to induce morphological defects in at least 20% of surviving embryos for each drug. The results showed that caffeine, IBMX, pentoxifyline and theophylline required ≤1 ng of drug; aminophylline and doxofylline required 3-5 ng of drug to induce the morphological defects in at least 20% of the embryos. However, diprophylline and etophylline didn’t cause morphological defects in 20% of the embryos even with the highest doses of 20 ng and 5 ng respectively.
Cardiac toxicity and Teratogenicity
The cardiovascular effects of methylxanthines in zebrafish embryos were more extensively evaluated. Zebrafish embryos were injected with the same drugs and doses (see Table 1) at 1-2 cell stage. Embryos were then analyzed for the structural and functional alterations.
Structural alteration
Assessment of the structural alteration of the heart was performed at 48 hpf. The embryos showing structural cardiac defect were termed cardiac phenotype and embryos with normal cardiac morphology were termed normal phenotype. The total percentage of embryos with cardiac phenotype was calculated for each of the compounds (Fig. 4a). Each of the embryos with cardiac phenotype was further assessed and graded based on the the criteria modified by the study of Panzica Kelly et al. [34]. The structural cardiac deformity in the embryos was scored as follows: 3 in case of normal heart morphology with slightly smaller ventricle, 2 if the atrium and ventricle were either compressed or severely enlarged and misshaped with no clear boundary between the atrium and ventricle and 1 if the atrium and ventricles were severely deficient, misshaped and not well defined.
Our results showed that with the exception of diprophylline and etophylline, all the other drugs induced cardiac phenotype in 20-40% of embryos with the highest dose. The minimum dose that caused at least 10% cardiac defects was 0.15 ng for IBMX, 0.25 ng for caffeine, pentoxifylline and theophylline,1 ng for doxofylline, 2.5 ng for aminophylline and 5 ng for etophylline. With diprophylline, the cardiac defect was present in less than 10% of the embryos even with the highest concentration of 20 ng. Further analysis of the embryos with cardiac phenotype showed that most of them had grade 2 cardiac abnormality. They had severely enlarged atrium and ventricles with distorted shape and not clearly defined border (Fig. 4c, d).
Evaluation of cardiovascular endpoints
In the embryos with structural cardiac defect, we also looked for several other cardiovascular endpoints at 48 hpf (Table 3). We observed that most of these embryos also had pericardial edema and decreased or absent blood circulation. Haemorrhage occurred only in few embryos injected with caffeine and theophylline, while thrombosis was absent in all the drug injected embryos.
To see the long term fate of the embryos with cardiovascular alterations, one concentration of each drug was injected in the BMP transgenic zebrafish embryos and tracked till 120 hpf. The embryos with structural cardiac defects either died or deteriorated further by 120 hpf.
Functional alteration
Heart rate in the normal looking embryos was measured at 48 hpf to assess the functional alteration of cardiac system. As methylxanthines are adenosine receptor antagonist, we expected to observe an increase in heart rate in the treated embryos if they were affected at functional level. Ten embryos were randomly selected for each concentration of each drug and the heart beat was counted. Embryos treated in sterile water were used as a negative control and metoprolol treated embryos were used as an internal control.
Our results showed that all the methylxanthines with the exception of doxofylline caused a significant increase in heart rate as compared to controls (Fig. 5). Doxofylline did not alter the heart rate whereas metoprolol significantly decreased the heart rate.
To see if the functional effect of these drugs in the embryos was a long lasting effect, the heart rate was tracked every 24 h from 48 hpf till 120 hpf in the BMP transgenic zebrafish embryos. We found that there was no significant difference in the heart rate in the treated and control embryos at 72, 96 and 120 hpf (Additional file 1).