Carbonic Anhydrase Inhibitors Induce Developmental Toxicity During Zebrafish Embryogenesis, Especially in the Inner Ear.

In vertebrates, carbonic anhydrases (CAs) play important roles in ion transport and pH regulation in many organs, including the eyes, kidneys, central nervous system, and inner ear. In aquatic organisms, the enzyme is inhibited by various chemicals present in the environment, such as heavy metals, pesticides, and pharmaceuticals. In this study, the effects of CA inhibitors, i.e., sulfonamides [ethoxyzolamide (EZA), acetazolamide (AZA), and dorzolamide (DZA)], on zebrafish embryogenesis were investigated. In embryos treated with the sulfonamides, abnormal development, such as smaller otoliths, an enlarged heart, an irregular pectoral fin, and aberrant swimming behavior, was observed. Especially, the development of otoliths and locomotor activity was severely affected by all the sulfonamides, and EZA was a consistently stronger inhibitor than AZA or DZA. In the embryos treated with EZA, inner ear hair cells containing several CA isoforms, which provide HCO3- to the endolymph for otolith calcification and maintain an appropriate pH there, were affected. Acridine orange/ethidium bromide staining indicated that the hair cell damage in the inner ear and pectral fin is due to apoptosis. Moreover, RNA measurement demonstrated that altered gene expression of cell cycle arrest- and apoptosis-related proteins p53, p21, p27, and Bcl-2 occurred even at 0.08 ppm with which normal development was observed. This finding suggests that a low concentration of EZA may affect embryogenesis via the apoptosis pathway. Thus, our findings demonstrated the importance of potential risk assessment of CA inhibition, especially regarding the formation of otoliths as a one of the most sensitive organs in embryogenesis.

Fluorinated benzenesulfonamide anticancer inhibitors of carbonic anhydrase IX exhibit lower toxic effects on zebrafish embryonic development than ethoxzolamide.

The toxic effects of two recently discovered inhibitors (VD12-09 and VD11-4-2) that selectively and with extraordinary strong, picomolar binding affinity to human carbonic anhydrase (CA) isoform IX were investigated on zebrafish embryonic development. CA IX has been recently introduced as an anticancer target since it is highly overexpressed in numerous human cancers but nearly absent in normal tissues. Morphological changes in zebrafish treated by the compounds were studied by light-field microscopy and histological analysis. Homology models of zebrafish CA II and CA IX were built to identify the conserved amino acid residues in the active site of zebrafish CAs. The toxicity studies here showed that the LC50 values at 120 hours post-fertilization (hpf) were 13 µM for VD12-09, 120 µM for VD11-4-2, and 9 µM for ethoxzolamide (EZA), a non-selective CA inhibitor commonly used as a drug in clinics. Thus, EZA was the most toxic of the three compounds. The zebrafish embryos exposed to LC50 doses of VD12-09 and VD11-4-2 showed fewer phenotypic abnormalities compared with the embryos exposed to the corresponding dose of EZA. Histochemical studies did not show any gross morphological changes in the embryos treated with VD12-09 and VD11-4-2 unlike EZA. The results of our study indicate that the compounds exhibited 10-fold lower toxicity and induced fewer side effects in zebrafish than EZA. Therefore, the exposure to VD11-4-2 and VD12-09 at concentrations below LC50 did not lead to deleterious effects on the zebrafish embryonic development and thus both inhibitors may be further developed as drugs.