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Background: Idiopathic intracranial hypertension (IIH) is a disease characterized by elevated intracranial pressure (ICP) and is a disease of young females. The first line pharmacological treatments include acetazolamide and topiramate and given the nature of IIH patients and the dosing regimen of these drugs, their effect on the endocrine system is important to evaluate. We aimed to assess the effects of acetazolamide and topiramate on steroid profiles in relevant endocrine tissues., Methods: Female Sprague Dawley rats received chronic clinically equivalent doses of acetazolamide or topiramate by oral gavage and were sacrificed in estrus. Tissue specific steroid profiles of lateral ventricle CP, 4th ventricle CP, CSF, serum, uterine horn and fundus, ovaries, adrenal glands and pituitary glands were assessed by quantitative targeted LC-MS/MS. We determined luteinizing hormone (LH) and follicle stimulating hormones (FSH) levels in paired serum by ELISA., Results: Topiramate increased the concentration of estradiol and decreased the concentration of DHEA in lateral choroid plexus. Moreover, it decreased the concentration of androstenediol in the pituitary gland. Topiramate increased serum LH. Acetazolamide decreased progesterone levels in serum and uterine fundus and increased corticosteroid levels in the adrenal glands., Conclusion: These results demonstrate that both acetazolamide and topiramate have endocrine disrupting effects in rats. Topiramate primarily targeted the choroid plexus and the pituitary gland while acetazolamide had broader systemic effects. Furthermore, topiramate predominantly targeted sex hormones, whereas acetazolamide widely affected all classes of hormones. A similar effect in humans has not yet been documented but these concerning findings warrants further investigations., Competing Interests: Declaration of competing interest The authors declare the following financial interests/personal relationships which may be considered as potential competing interests: Connar Westgate reports financial support was provided by Lundbeck Foundation. Rigmor Jensen reports financial support was provided by Lundbeck Foundation. If there are other authors, they declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 The Author(s). Published by Elsevier Inc. All rights reserved.)

The increasing presence of anthropogenic contaminants in the environment may constitute a challenge to non-target biota, considering that most contaminants can exert deleterious effects. Salicylic acid (SA) is a non-steroid anti-inflammatory drug (NSAID) which exerts its activity by inhibiting the enzyme cyclooxygenase (COX). Another class of drugs is that of the diuretics, in which acetazolamide (ACZ) is included. This pharmaceutical acts by inhibiting carbonic anhydrase (CA), a key enzyme in acid-base homeostasis, regulation of pH, being also responsible for the bio-availability of Ca 2+ for shell biomineralization processes. In this work, we evaluated the chronic (28-day) ecotoxicological effects resulting from the exposures to SA and ACZ (alone, and in combination) on individuals of the marine mussel species Mytillus spp., using enzymatic (catalase (CAT), glutathione S-transferases (GSTs), COX and CA), non-enzymatic (lipid peroxidation, TBARS levels) and morphological and physiological (shell hardness, shell index and feeding behaviour) biomarkers. Exposure to ACZ and SA did not cause significant alterations in CAT and GSTs activities, and in TBARS levels. In terms of CA, this enzyme was inhibited by the highest concentration of ACZ in gills of exposed animals, but no effects occurred in the mantle tissue. The activity of COX was not altered after exposure to the single chemicals. However, animals exposed to the mixture of ACZ and SA evidenced a significant inhibition of COX activity. Morphological and physiological processes (namely, feeding, shell index, and shell hardness) were not affected by the here tested pharmaceutical drugs. Considering the general absence of adverse effects, further studies are needed to fully evaluate the effects of these pharmaceutical drugs on alternative biochemical and physiological pathways., (Copyright © 2021 Elsevier Ltd. All rights reserved.)

Pharmaceutical drugs are among the most used chemicals for human and veterinary medicines, aquaculture and agriculture. Pharmaceuticals are environmentally persistent, biologically active molecules, thereby having the potential to exert biological effects on non-target species. Among the most used pharmaceuticals, one may find salicylic acid (SA), a non-steroid anti-inflammatory drug (NSAID) that acts by inhibiting the enzymes cyclooxigenases; it is also possible to identify acetazolamide (ACZ), a diuretic that acts by inhibiting the activity of carbonic anhydrase (CA). In this work, the effects of both single and combined effects of these drugs were assessed on the marine gastropod Phorcus lineatus, by measuring key enzymatic activities, namely carbonic anhydrase (CA) and cyclooxygenase (COX), under two different exposure periods (14 and 28 days). We observed no straightforward pattern of enzymatic response in all treatments of both pharmaceuticals, on both analyzed tissues (gut and gills), and for both exposure regimes. We assume that this species is not responsive to the hereby tested pharmaceuticals, a finding that may be due to general mechanisms of response to adverse conditions, such as reduction of metabolism, of heart rate, of filtration rates, and to the increase production of mucus. All these functional adaptations can mitigate the deleterious effects caused by adverse conditions, without triggering biochemical responses. In conclusion, the species P. lineatus seems not to be sensitive in terms of these specific enzymatic pathways to these contaminants, under the adopted conditions.

Pharmaceutical drugs are among the most used chemicals, for human and veterinary medicines, aquaculture and agriculture. Pharmaceuticals are biologically active molecules, having also environmental persistence, thereby exerting biological effects on non-target species. Among the most used pharmaceuticals, one may find salicylic acid (SA), a non-steroid anti-inflammatory drugs (NSAIDs), and acetazolamide (ACZ), a diuretic drug that acts by inhibiting the activity of carbonic anhydrase (CA). In this work, single and combined effects of SA and ACZ were assessed in the aquatic macrophyte Lemna gibba L., focusing on physiological parameters, namely photosynthetic pigments, (chlorophyll a, b and total (Chl a, b and TChl) as well as carotenoids (Car)). In addition, chemical biomarkers, namely, glutathione S-transferases (GSTs), catalase (CAT) and carbonic anhydrase (CA) activities, were also determined. The highest concentrations of ACZ, caused a decrease in the contents of all chlorophylls; this effect was however reverted by SA exposure. Both ACZ and SA levels caused a decrease in CA activity. Nevertheless, when in combination, this inhibition was not observed in plants exposed to the lowest concentration of these drugs. In conclusion, both pharmaceuticals have the capacity to cause alterations in L. gibba enzymatic activity and photosynthetic pigments content. Additionally, SA seems to exert a protective effect on this species against deleterious effects caused by ACZ., (Copyright © 2020. Published by Elsevier B.V.)

Vancomycin-resistant enterococci (VRE) are the second leading cause of hospital-acquired infections (HAIs) attributed to a drug-resistant bacterium in the United States, and resistance to the frontline treatments is well documented. To combat VRE, we have repurposed the FDA-approved carbonic anhydrase drug acetazolamide to design potent antienterococcal agents. Through structure-activity relationship optimization we have arrived at two leads possessing improved potency against clinical VRE strains from MIC = 2 μg/mL (acetazolamide) to MIC = 0.007 μg/mL ( 22 ) and 1 μg/mL ( 26 ). Physicochemical properties were modified to design leads that have either high oral bioavailability to treat systemic infections or low intestinal permeability to treat VRE infections in the gastrointestinal tract. Our data suggest the intracellular targets for the molecules are putative α-carbonic and γ-carbonic anhydrases, and homology modeling and molecular dynamics simulations were performed. Together, this study presents potential anti-VRE therapeutic options to provide alternatives for problematic VRE infections.

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 HCO 3 - 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.

In the present work the antiglaucoma drug, acetazolamide, was formulated as an ion induced nanoemulsion-based in situ gel for ocular delivery aiming a sustained drug release and an improved therapeutic efficacy. Different acetazolamide loaded nanoemulsion formulations were prepared using peanut oil, tween 80 and/or cremophor EL as surfactant in addition to transcutol P or propylene glycol as cosurfactant. Based on physicochemical characterization, the nanoemulsion formulation containing mixed surfactants and transcutol P was selected to be incorporated into ion induced in situ gelling systems composed of gellan gum alone and in combination with xanthan gum, HPMC or carbopol. The nanoemulsion based in situ gels showed a significantly sustained drug release in comparison to the nanoemulsion. Gellan/xanthan and gellan/HPMC possessed good stability at all studied temperatures, but gellan/carbopol showed partial drug precipitation upon storage and was therefore excluded from the study. Gellan/xanthan and gellan/HPMC showed higher therapeutic efficacy and more prolonged intraocular pressure lowering effect relative to that of commercial eye drops and oral tablet. Gellan/xanthan showed superiority over gellan/HPMC in all studied parameters and is thus considered as a promising mucoadhesive nanoemulsion-based ion induced in situ gelling formula for topical administration of acetazolamide., (Copyright © 2017. Published by Elsevier B.V.)

Reactions of pial vessels to hypercapnia were studied in Wistar rats one week after global cerebral ischemia. In ischemic rats, the responsiveness of pial vessels to hypercapnia was diminished, which promoted a decrease in cerebral perfusion reserve. Changes in vascular responsiveness in the arterial and venous subdivisions of the vascular bed were observed and probably resulted from ischemia-provoked down-regulation of the vascular tone.

Large oral doses of ACZ lower the intraocular pressure (IOP), but usually lead to a multitude of systemic side effects, including gastrointestinal upset. The present study was undertaken to evaluate the effect of ACZ on the histological structure of rat duodenal mucosa and to assess a possible protective role of the complex formation of ACZ with HP-β-CD, either separately or in combination with a third compound, on the gut epithelial layer by histological and ultrastructural examinations of sections of rat duodenum exposed to ACZ or its formulations. In addition, the transport process of ACZ and its binary or ternary complexes across the duodenal mucosa by means of the single-pass intestinal perfusion (SPIP) method in rats was evaluated. Evidence was found that ACZ alters intestinal permeability and induces damage to the rat small intestine. In contrast, ACZ-induced intestinal injury may be abrogated by ACZ complexation. In addition, the complexation of ACZ with HP-β-CD, alone or in combination with a third compound, facilitated significant levels of ACZ uptake across the rat duodenal segment. Ternary complexes of ACZ with HP-β-CD in combination with TEA (triethanolamine) or calcium ions were found to provide an excellent approach that enabled an increased apparent permeability of ACZ across the duodenal epithelium, with a concomitant ability to preserve the integrity of the gut epithelium from ACZ-induced injury. These results could be useful for the design and development of novel ACZ formulations that can reduce GI toxicity, while still maintaining their essential therapeutic efficacies., (Copyright © 2014. Published by Elsevier B.V.)

The present investigation was aimed to develop topically effective acetazolamide loaded poly(propylene imine) dendrimer nanoarchitectures and evaluate their intraocular pressure lowering potential. The 5.0G PPI dendrimers were synthesized using ethylendiamine as dendrimer core through divergent approach and characterized and loaded with acetazolamide (ACZ). The developed dendrimeric formulations were characterized for size, loading efficiency. The surface morphology of dendrimer was studied by Transmission Electron Microscopy. The developed dendrimer formulations were evaluated for hemolytic toxicity, ocular irritation index and intra ocular pressure reduction using normotensive adult male New Zealand albino rabbits as in vivo model. The maximum drug entrapment efficiency was found to be 56±2.3%. The in vitro release data of ACZ-5.0G PPI dendrimers showed sustained release of ACZ which was found to be 83.5±1.8 and 80.4±1.6% in phosphate buffer saline (pH 7.4) and simulated tear fluid (pH 7.4), respectively in 24h. Ocular irritancy, ocular residence time and intraocular pressure lowering effect were performed. The study revealed that in lower concentrations the aqueous solutions of dendrimer formulations were found to be weakly irritant to the eyes. The sustained and prolonged reduction in intraocular pressure suggested that drug entrapped in dendrimers can be used for higher retention in ocular cul-de sac. Further, the PPI dendrimer based formulation seems to enhance the ocular drug residence time and exhibits better intraocular pressure lowering effect for glaucoma treatment, more safely, both in vitro and in vivo., (Copyright © 2013 Elsevier B.V. All rights reserved.)

Purpose: To evaluate the performance of 6-O-Lauryl-l-ascorbic acid nanostructures (coagels) as vehicles for acetazolamide (AZM) in ophthalmic administration by in vitro and in vivo experimental tests., Methods: The systems of coagel + AZM were evaluated in terms of their in vitro release (dialysis membrane), permeability (isolated cornea), pharmacological effectiveness [intraocular pressure (IOP)-reduction in normotensive rabbits], and potential irritant effects., Results: The results concerning AZM permeation were better when vehiculized in coagels compared with ringer solution, which was evident from the AZM steady-state flux and P(app) values (J=1.43 μg/min and P(app)=3.04 cm.s(1)). As a consequence of this increase in permeation, the coagel-AZMs were more effective in lowering the IOP, according to the results obtained from the in vivo assays. Coagels loaded with 0.4% (W/W) of AZM showed a higher hypotensive effect in rabbits compared with the commercial formulation AZOPT(®) (brinzolamide 1%), mainly due to the prolonged effect of the former. In all cases, the intensity of irritation was time dependent. The sodium lauryl sulphate solution (2%) used as a positive control produced serious injury 30 min postadministration. This effect caused irritation, which decreased slowly and even at 180 min, the discomfort was still considerable. However, in the case of coagels, a mild-to-moderate effect was observed., Conclusions: The incorporation of AZM in coagels seems to improve the ocular bioavailability of this drug. Coagel-AZM 0.4% showed a higher hypotensive effect, with a mild-to-moderate irritant effect. These systems could be administrated in human beings, although more detailed studies still need to be carried out.

Skeletal changes induced by treatment of pregnant rats with four potent teratogens, busulfan, acetazolamide, vitamin A palmitate, and ketoconazole, were evaluated using Alizarin Red S and Alcian Blue double-staining to investigate the relationship between drug-induced skeletal malformations and cartilaginous changes in the fetuses. Pregnant rats (N = 8/group) were treated once or twice between gestation days (GDs) 10 to 13 with busulfan at doses of 3, 10, or 30 mg/kg; acetazolamide at 200, 400, or 800 mg/kg; vitamin A palmitate at 100,000, 300,000, or 1,000,000 IU/kg; or ketoconazole at doses of 10, 30, or 100 mg/kg. Uterine evaluations and fetal external and skeletal examinations were conducted on GD 20. Marked skeletal abnormalities in ribs and hand/forelimb bones such as absent/ short/bent ribs, fused rib cartilage, absent/fused forepaw phalanx, and misshapen carpal bones were induced at the mid- and high-doses of busulfan and acetazolamide and at the high-dose of vitamin A palmitate and ketoconazole. Increased incidences of discontinuous rib cartilage (DRC) and fused carpal bone (FCB) were observed from the low- or mid-dose in the busulfan and acetazolamide groups, and incidences of FCB were increased from the mid-dose in the vitamin A palmitate and ketoconazole groups. Therefore, DRC and FCB were detected at lower doses than those at which ribs and hand/forelimb malformations were observed in the four potent teratogens.

Embryotoxicity of glycol ethers is caused by their alkoxyacetic acid metabolites, but the mechanism underlying the embryotoxicity of these acid metabolites is so far not known. The present study investigates a possible mechanism underlying the embryotoxicity of glycol ether alkoxyacetic acid metabolites using the methoxyacetic acid (MAA) metabolite of ethylene glycol monomethyl ether as the model compound. The results obtained demonstrate an MAA-induced decrease of the intracellular pH (pH(i)) of embryonic BALB/c-3T3 cells as well as of embryonic stem (ES)-D3 cells, at concentrations that affect ES-D3 cell differentiation. These results suggest a mechanism for MAA-mediated embryotoxicity similar to the mechanism of embryotoxicity of the drugs valproic acid and acetazolamide (ACZ), known to decrease the pH(i)in vivo, and therefore used as positive controls. The embryotoxic alkoxyacetic acid metabolites ethoxyacetic acid, butoxyacetic acid and phenoxyacetic acid also caused an intracellular acidification of BALB/c-3T3 cells at concentrations that are known to inhibit ES-D3 cell differentiation. Two other embryotoxic compounds, all-trans-retinoic acid and 5-fluorouracil, did not decrease the pH(i) of embryonic cells at concentrations that affect ES-D3 cell differentiation, pointing at a different mechanism of embryotoxicity of these compounds. MAA and ACZ induced a concentration-dependent inhibition of ES-D3 cell differentiation, which was enhanced by amiloride, an inhibitor of the Na(+)/H(+)-antiporter, corroborating an important role of the pH(i) in the embryotoxic mechanism of both compounds. Together, the results presented indicate that a decrease of the pH(i) may be the mechanism of embryotoxicity of the alkoxyacetic acid metabolites of the glycol ethers., ((c) 2010 Elsevier Inc. All rights reserved.)

Background: A variety of drugs, environmental chemicals, and physical agents induce a common limb malformation in the offspring of pregnant mice exposed on day 9 of gestation. This malformation, postaxial, right-sided forelimb ectrodactyly, is thought to arise via an alteration of hedgehog signaling., Methods: We have studied two of these teratogens, acetazolamide and cadmium, using the technique of microarray analysis of limb bud ectoderm and mesoderm to search for changes in gene expression that could indicate a common pathway to postaxial limb reduction., Results: Results indicated a generalized up-regulation of gene expression after exposure to acetazolamide but a generalized down-regulation due to cadmium exposure. An intriguing observation was a cadmium-induced reduction of Mt1 and Mt2 expression in the limb bud mesoderm indicating a lowering of embryonic zinc., Conclusions: We propose that these two teratogens and others (valproic acid and ethanol) lower sonic hedgehog signaling by perturbation of zinc function in the sonic hedgehog protein.

This aim of this study was to investigate the biocompatibility of two experimental acetazolamide (AZ)-based pastes in the subcutaneous tissue of rats. Both pastes contained AZ as the main component in similar concentration. The vehicle in experimental paste 1 was saline, while experimental paste 2 was prepared with propylene glycol. Sixty polyethylene tubes were sealed at one end with gutta-percha (GP), which served as a control. Half of the tubes were filled with paste 1 and half with paste 2. The tubes were implanted in the subcutaneous tissue of 15 rats, being 4 tubes for each animal. The animals were killed 7, 15 and 45 days after surgery and the specimens were processed in laboratory. The histological sections were stained with hematoxylin and eosin and were analyzed by light microscopy. Scores were assigned to level of inflammatory process: 1- none; 2- mild; 3- moderate; 4- severe. The data were analyzed statistically by the Kruskal-Wallis test (p< or =0.05). Paste 1 produced an inflammatory process at 7 days. However, the intensity of this inflammation decreased with time and was nearly absent at 45 days. No statistically significant difference (p>0.05) was observed between the control (GP) and paste 1. However, paste 2 produced inflammatory response at all study periods and differed significantly (p<0.05) from the control. In conclusion, in the present study, the experimental AZ-based paste 1 was considered as biocompatible as the control matrial (GP), while experimental paste 2 was irritating to rat subcutaneous tissue.

The Cl(-)/anion exchanger pendrin (SLC26A4) is expressed on the apical side of renal non-type A intercalated cells. The abundance of pendrin is reduced during metabolic acidosis induced by oral NH(4)Cl loading. More recently, it has been shown that pendrin expression is increased during conditions associated with decreased urinary Cl(-) excretion and decreased upon Cl(-) loading. Hence, it is unclear if pendrin regulation during NH(4)Cl-induced acidosis is primarily due the Cl(-) load or acidosis. Therefore, we treated mice to increase urinary acidification, induce metabolic acidosis, or provide an oral Cl(-) load and examined the systemic acid-base status, urinary acidification, urinary Cl(-) excretion, and pendrin abundance in the kidney. NaCl or NH(4)Cl increased urinary Cl(-) excretion, whereas (NH(4))(2)SO(4), Na(2)SO(4), and acetazolamide treatments decreased urinary Cl(-) excretion. NH(4)Cl, (NH(4))(2)SO(4), and acetazolamide caused metabolic acidosis and stimulated urinary net acid excretion. Pendrin expression was reduced under NaCl, NH(4)Cl, and (NH(4))(2)SO(4) loading and increased with the other treatments. (NH(4))(2)SO(4) and acetazolamide treatments reduced the relative number of pendrin-expressing cells in the collecting duct. In a second series, animals were kept for 1 and 2 wk on a low-protein (20%) diet or a high-protein (50%) diet. The high-protein diet slightly increased urinary Cl(-) excretion and strongly stimulated net acid excretion but did not alter pendrin expression. Thus, pendrin expression is primarily correlated with urinary Cl(-) excretion but not blood Cl(-). However, metabolic acidosis caused by acetazolamide or (NH(4))(2)SO(4) loading prevented the increase or even reduced pendrin expression despite low urinary Cl(-) excretion, suggesting an independent regulation by acid-base status.

To test for environmental persistence in order to determine the potential of carbonic anhydrase inhibitors as larvicides, the decomposition and degradation of samples containing methazolamide (MTZ) and acetazolamide (ACZ) in aqueous solution were monitored under different conditions. Additionally, nontarget species impact was assessed in an acute toxicity test using sheepshead minnow (Cyprinodon variegatus). The fish were exposed for 120 h to 10(-3) and 10(-4) M each compound in replicate seawater tanks. In the high-MTZ treatment, all fish died within 48 h, while mortality in the low-MTZ treatment was 27% at 120 h. In the high-ACZ treatment mortality reached 83% at 120 h. We observed no mortality for the lowest dose of ACZ. Tissue samples were collected from the fish to investigate absorption of the compounds. In the gills, MTZ concentrations were around 40 microg g(-1) and ACZ reached concentrations up to 80 microg g(-1). Liver concentrations were low for MTZ probably due to metabolism.

Aspirin (ASA), an irreversible cyclooxygenase (COX) inhibitor, induces ventricular septal defect (VSD) and diaphragmatic hernia (DH) in rat fetuses when administered on gestation days (GDs) 9-10, a critical period for cardiovascular (CV) and midline development. Evaluation of a spectrum of nonsteroidal antiinflammatory drugs (NSAIDs; reversible COX inhibitors) showed that while some NSAIDs induced VSD in rats, none of the NSAIDs evaluated produced DH. In addition to inhibiting COX, ASA also inhibits carbonic anhydrase. The purpose of this study was to determine whether concurrent inhibition of COX and carbonic anhydrase would produce a teratogenic profile that includes both VSD and DH. To inhibit both COX and carbonic anhydrase, ibuprofen (COX inhibitor) and acetazolamide (carbonic anhydrase inhibitor) were coadministered on GDs 9-10. Groups of 20 female Crl:CD(SD)IGS BR rats were given either 300 mg kg(-1) day(-1) ibuprofen, 1000 mg kg(-1) day(-1) acetazolamide, or both (combination of ibuprofen and acetazolamide). Fetuses were evaluated on GD 21 for external and visceral development. Ibuprofen induced VSD in 3.7% of fetuses per litter; no defects in appendicular skeletal development were noted. Acetazolamide induced VSD in 5.9% of the fetuses per litter and appendicular defects in 41% of the fetuses per litter. Coadministration of ibuprofen and acetazolamide produced VSD in 18.7% of the fetuses per litter and appendicular defects in 77% of the fetuses per litter; however, there were no DH. Therefore, while concurrent inhibition of COX and carbonic anhydrase did not produce DH, potentiation was noted for the induction of VSD and appendicular anomalies.

Purpose: NH4Cl gavage in the neonatal rat produces a metabolic acidosis-induced retinopathy which serves as a model for retinopathy of prematurity (ROP). Acetazolamide induces a metabolic acidosis via an alternative biochemical mechanism (bicarbonate loss versus hydrogen ion load). In the present study, the following hypothesis was tested: acetazolamide-induced acidosis is associated with preretinal neovascularization in the neonatal rat., Methods: All studies used newborn Sprague-Dawley rats raised in expanded litters of 25. Arterial blood pH was measured to determine the level of acidosis induced by intraperitoneal (IP) acetazolamide (50 or 200 mg/kg) or saline. In a separate retinopathy study, newborn rats (n = 75) were randomized to either IP acetazolamide, 50 mg/kg (low-dose), or IP saline twice daily from days 2 to 7. After 5 days of recovery, retinal vasculature was assessed using ADPase staining and light microscopy. The presence and severity (clock hours) of neovascularization were assessed by three masked observers. In an additional retinopathy study, newborn rats (n = 100) were randomized to either IP acetazolamide, 200 mg/kg (high-dose), or IP saline twice daily from days 2 to 7. After 5 days of recovery, the retinas were similarly analyzed., Results: Neovascularization occurred in 59% of rats receiving high-dose acetazolamide (200 mg/kg). High-dose acetazolamide produced a severe acidosis (pH 7.13 +/- 0.06) during drug delivery. Low-dose acetazolamide (50 mg/kg) produced a pH (7.22 +/- 0.07) that was intermediate between high-dose (200 mg/kg) acetazolamide (P < 0.001) and saline controls (7.42 +/- 0.06, P < 0.001); however, neither low-dose acetazolamide nor saline induced preretinal neovascularization., Conclusions: Acidosis induced by high-dose acetazolamide, independent of hyperoxemia or hypoxemia, is associated with preretinal neovascularization in the neonatal rat. Induction of neovascularization appears to depend on a critical threshold of acidosis severity. This study further supports a proposed independent role for acidosis in the pathogenesis of ROP.

Between days 9.5 and 10, the forelimb buds of developing murine embryos progress from stage 1 which are just beginning to express shh and whose posterior mesoderm has only weak polarizing activity to stage 2 limbs with a distinguishable shh expression domain and full polarizing activity. We find that exposure on day 9.5 to teratogens that induce the loss of posterior skeletal elements disrupts the polarizing activity of the stage 2 postaxial mesoderm and polarizing activity is not subsequently restored. The ontogeny of expression of the mesodermal markers shh, ptc, bmp2, and hoxd-12 and 13, as well as the ectodermal markers wnt7a, fgf4, fgf8, cx43, and p21 occurred normally in day 9.5 teratogen-exposed limb buds. At stage 3, the treated limb apical ectodermal ridge usually possessed no detectable abnormalities, but with continued outgrowth postaxial deficiencies became evident. Recombining control, stage matched limb bud ectoderm with treated mesoderm prior to ZPA grafting restored the duplicating activity of treated ZPA tissue. We conclude that in addition to shh an early ectoderm-dependent signal is required for the establishment of the mouse ZPA and that this factor is dependent on the posterior ectoderm.

A small percentage of C57BL/6 mice spontaneously develop focal collections of neurons in the molecular layer of the cerebral neocortex. Usually only one "ectopia" is present in each affected brain. Studies in other mouse strains have shown that these ectopias occur before birth, probably because of a breach in the superficial glial membrane during neuronal migration. The ectopias are heritable and are caused by multiple genes. C57BL/6J mice exposed prenatally to acetazolamide, a carbonic anhydrase-specific inhibitor and teratogen, develop an increased frequency of limb malformations, especially in the right forelimb. In the present study, we hypothesized that the prevalence and severity of ectopias would be increased in acetazolamide-exposed mice because carbonic anhydrase plays a key role in brain development. Further, we wanted to determine whether there was a correlation between the side of limb deformity and the hemisphere containing an ectopia. Thus, we injected C57BL/6J time-mated mice intraperitoneally on embryonic day 9 with either sodium acetazolamide (750 mg/kg) or water. Histological analysis of the brains from 105 acetazolamide-exposed offspring and 89 control offspring revealed no difference in the overall prevalence of cerebrocortical ectopias between the acetazolamide and control groups: 34% of the acetazolamide-exposed and 28% of the control mice had ectopias. There was, however, a striking difference in the shape and size of ectopias: 67% of the ectopias were large in the acetazolamide-exposed group in comparison to 32% in controls. The acetazolamide-exposed offspring also were more likely to have multiple ectopias. Thus, there may be a genetic predisposition for developing ectopias in some mouse strains, but epigenetic factors such as prenatal exposure to acetazolamide can influence their severity., (Copyright 1999 Wiley-Liss, Inc.)

Postaxial forelimb ectrodactyly induced by acetazolamide given on Day 9.5 of murine gestation is thought to be mediated by reduced intracellular pH (pHi) within the limb bud. Coadministration of amiloride increases the incidence and severity of acetazolamide-induced forelimb malformations and further reduces limb bud pHi. These findings were hypothesized to be attributable to the action of amiloride as an inhibitor of Na+/H+ exchangers (NHEs), plasma membrane-localized proteins involved in the maintenance of cellular pH homeostasis. Here, we explored this hypothesis further by coadministering with acetazolamide, amiloride, or analogs known to preferentially inhibit NHEs 5-(N-methyl-N-isobutyl)-amiloride, 5-(N, N-hexamethylene)-amiloride, 5-(N, N-dimethyl)-amiloride, and 5-(N-ethyl-N-isopropyl)-amiloride or amiloride-sensitive Na+ channels (benzamil). The coadministration of either amiloride, benzamil, 5-(N, N-dimethyl)-amiloride, 5-(N-ethyl-N-isopropyl)-amiloride, or 5-(N-methyl-N-isobutyl)-amiloride all dose responsively increased the frequency and severity of forelimb malformations compared to acetazolamide alone. None of the analogs given alone induced forelimb ectrodactyly. The data are consistent with the original hypothesis that the exacerbation of acetazolamide teratogenesis is due to NHE inhibition. Surprisingly, benzamil was the most potent potentiator of acetazolamide teratogenesis. This result strongly suggests that amiloride-sensitive Na+ channels are also present within the murine embryo and are likely to play a role in pHi homeostasis.

Acetazolamide, a carbonic anhydrase inhibitor, has been shown effective in the treatment of cystoid macular edema; however, chronic use of the drug is limited by its serious systemic side effects. Intraocular administration can eliminate these systemic complications. We evaluated vitreoretinal toxicity after intravitreal injection of acetazolamide in the rabbit eye. The right eye of each rabbit received a single acetzolamide injection; the left eye received balanced salt solution and served as a control. The effect of the drug was evaluated by clinical observation, electroretinography, and histopathologic study. Intravitreal injection of up to 0.5 mg acetazolamide did not cause vitreoretinal toxicity. Injection of 1 mg or higher doses depressed the b-wave amplitude of electroretinograms and damaged the outer segments of the photoreceptors, as determined by light and electron microscopy.

Renal papillary cytoplasmic granularity (RPCG) induced by carbonic anhydrase inhibitors (CAIs) in rats is characterized by the accumulation of dense secondary lysosomes in epithelial, endothelial, and interstitial cells and may be related to drug-induced potassium depletion. Female Sprague-Dawley rats were given the CAI, acetazolamide, by gavage. Half were supplemented with 1% potassium chloride in the drinking water. Two treated groups were allowed to recover for 1 or 2 mo. Potassium supplementation inhibited RPCG by 80% but produced little amelioration of the mild 13% decrease in serum potassium induced by 200 mg/kg/day acetazolamide for 28 days. Acetazolamide-induced RPCG is reversible because 1- and 2-mo recovery periods decreased the incidence by 75% and 80%, respectively. The results support the hypothesis that RPCG is related to potassium depletion secondary to carbonic anhydrase inhibition. Because supplementation of potassium chloride had little effect on serum potassium, these data suggest that depletion of renal medullary potassium content is important in the pathogenesis of RPCG as previously suggested by others. Other types of diuretics that produce hypokalemia as a side effect may not deplete medullary potassium since RPCG has not been reported in humans or animals given these drugs.

Penetrance or the frequency of embryos with any degree of forelimb ectrodactyly is the usual method to describe the forelimb ectrodactyly response of mouse embryos to acetazolamide. A digit score for number of small or absent digits for the separate right and left forelimb response to acetazolamide provides a measure of expressivity or the severity of response. We examine the relationship between expressivity and penetrance using right and left forelimb data from a previously reported dose-response analysis of the C57BL/6J and WB/ReJ strains to acetazolamide. The data show that expressivity and penetrance are highly correlated for the separate right and left forelimbs for both strains. In C57BL/6J, the dose-response analyses of both expressivity and penetrance of the separate right and left forelimbs demonstrate a teratogenic gradient, decreasing from right to left, that depends on the symmetrical ectrodactyly response of the right and left forelimbs. In WB/ReJ, the right forelimb is also more sensitive than the left, but the dose-response analyses of both penetrance and expressivity show the two forelimbs are asymmetrical in their ectrodactyly response and that there is not a simple teratogenic gradient in this strain. In WB/ReJ, the left forelimb is resistant at even the highest non-lethal doses. The high correlation between expressivity and penetrance for the separate forelimbs of both C57BL/6J and WB/ReJ suggests that this right-left difference between the two strains may not be a property of the limbs themselves but may be an intrinsic genetic difference between the two types of embryos perhaps in the amount of teratogen to which the embryos are exposed.(ABSTRACT TRUNCATED AT 250 WORDS)

Pregnant SWV mice were treated on day 9 of gestation (PC) with 50 mg/kg of caffeine (CAFF), 200 mg/kg (LD) or 1000 mg/kg (HD) of acetazolamide (ACZM), or a combination of both agents, or on day 8 PC with both agents (ACZM + CAFF). Untreated (UNTD) and vehicle-treated (VEH) groups served as controls. The SWV strain is widely reported to be resistant to ACZM; it was resistant to ACZM or CAFF + ACZM when treated on day 9 of gestation, but a significant frequency of malformations, primarily exencephaly, was produced by ACZM + CAFF on day 8 PC. This study provides evidence that ACZM, coupled with a subteratogenic dose of caffeine can produce abnormalities in the "resistant" SWV mice, using the endpoint of exencephaly on day 8 of gestation. The mean number of ossified caudal vertebrae in day-9 treatments and ossified cervical vertebral centra in day-8 treatments were reduced. The frequency of ossification of the first cervical vertebra (C1) was reduced from 93% in UNTD to 39% in HD-ACZM day 9 PC and 69% in HD-ACZM + CAFF day 9 PC groups, and was also significantly reduced in the HD-ACZM + CAFF day-8 treated group.

These experiments more accurately define the effects of the combination acetazolamide-amiloride or a single dose of dimethadione (DMO), the active metabolite of trimethadione, on the development of the ureter. When acetazolamide-amiloride was administered in C57BL/6NCrlBR mice on day 9, 9.5, or 10 of gestation (plug = day 0) a second ureter was formed, anterior to the original ureter, inducing a second kidney. The second ureter then fails to make a connection with the developing bladder and remains attached to the mesonephric duct. The mesonephric duct becomes the vas deferens in the male and deteriorates completely in the female leading to either a restricted ureter or a blocked ureter depending on the sex of the fetus. Administration of a single dose of DMO between gestational day 9 and 10.3 produced both renal agenesis and ureters of varying lengths. Some ureters were of normal length with a tuft of one or two nephrons at their tip, while others were one half or one quarter of their normal length. In some instances the ureter was completely absent. The reason for this strong effect on the ureter is unknown.

In order to evaluate the teratogenic potential of acetazolamide in rabbits, three groups of 18 artificially inseminated females were treated orally with 50, 100, or 150 mg/kg/day of acetazolamide on days 6-18 of gestation. These doses induced maternal acidosis and electrolyte changes, consistent with those reported in rats and considered to be a result of carbonic anhydrase inhibition, as well as reductions in maternal body weight gain. At cesarean sections, average fetal body weights in the acetazolamide groups were dose-dependently decreased compared with controls. There were no effects of acetazolamide on embryonic survival or external morphology of live fetuses. In the fetal skeletal examination, thoracic and lumbar vertebral malformations occurred in 0.7%, 3.9%, and 6.1% of fetuses in the 50, 100, and 150 mg/kg/day groups, respectively, compared with none in the control group. In addition, missing vertebra was seen in a small number of fetuses in the 100 and 150 mg/kg/day groups. These axial skeletal malformations were, in some cases, associated with costal malformations. These results indicate that acetazolamide at maternotoxic doses can produce axial skeletal malformations in the rabbit.

In many empirical analyses, the response of interest is categorical with an ordinal scale attached. Many investigators prefer to formulate a linear model, assigning scores to each category of the ordinal response and treating it as continuous. When the covariates are categorical, Haber (1985, Computational Statistics and Data Analysis 3, 1-10) has developed a method to obtain maximum likelihood (ML) estimates of the parameters of the linear model using Lagrange multipliers. However, when the covariates are continuous, the only method we found in the literature is ordinary least squares (OLS), performed under the assumption of homogeneous variance. The OLS estimates are unbiased and consistent but, since variance homogeneity is violated, the OLS estimates of variance can be biased and may not be consistent. We discuss a variance estimate (White, 1980, Econometrica 48, 817-838) that is consistent for the true variance of the OLS parameter estimates. The possible bias encountered by using the naive OLS variance estimate is discussed. An estimated generalized least squares (EGLS) estimator is proposed and its efficiency relative to OLS is discussed. Finally, an empirical comparison of OLS, EGLS, and ML estimators is made.

The carbonic anhydrase inhibitors, acetazolamide and MK-0927, were given by oral route to male Sprague-Dawley rats at 200 mg/kg/day and 25 mg/kg/day, respectively, for up to 4 weeks. Sequential necropsies were performed and urinary bladders were examined by light microscopy (LM), scanning electron microscopy (SEM), and transmission electron microscopy (TEM). Similar urinary bladder changes were seen with both compounds. SEM evidenced slight multifocal urothelial changes consisting of cell swelling, dissociation, degeneration, and exfoliation after 3 and 5 days of treatment. After 2 and 4 weeks of treatment, elevated or leafy microridges on the luminal cell surfaces were seen together with foci of swollen cells. After a 2-month-recovery-period, the urothelial surfaces were normal. LM and TEM showed multifocal vacuolation of the urothelium associated with inflammation of the underlying lamina propria after 3 and 5 days of treatment. Cellular hypertrophy and hyperplasia of the transitional epithelium was seen after a 5-day treatment, persisted without increasing severity after 2 and 4 weeks of treatment, and totally regressed after the recovery period. It was concluded that, in the rat urinary bladder, oral administration of acetazolamide and MK-0927 induced early degeneration and inflammation followed by epithelial regeneration, resulting in a reversible hyperplasia of the transitional epithelium.

This study assesses the relative contributions of dam and conceptus to sensitivity to acetazolamide teratogenesis in an in vivo mouse model. Reciprocal F1 outcrosses were made between mice resistant to the teratogenic effects of acetazolamide (SWV) and the susceptible C57BL/6JBk line. Female F1 progeny were backcrossed either to C57BL or to SWV males. The F1 outcross resulted in identical fetuses developing in genetically different uterine environments. In the backcrosses, genetically identical hybrid dams were gestating genetically different populations of segregating fetuses. In both F1 outcrosses and backcrosses, as well as in pure-line controls, dams were treated on day 9 of gestation by subcutaneous injection of acetazolamide (ACZM) at a dose level of 1500 mg/kg, or identical volume of the vehicle. The principal abnormality seen was right front limb ectrodactyly. Among pure-line matings, SWV produced no ectrodactyly, but 68% of C57BL showed this defect. Only two cases were seen in outcross of C57BL females to SWV males, and one litter produced three ectrodactylous fetuses in the reciprocal cross. A highly significant increase in ectrodactyly was seen in treated backcrosses to C57BL relative to backcrosses to SWV. Thus, a litter in which most of the genes segregating are from a susceptible line will show major increases in susceptibility, despite being in a heterotic and somewhat resistant uterine environment, whereas the susceptible C57BL uterine environment does not result in significant abnormality in heterozygous resistant fetuses. These results unequivocally demonstrate that it is the genotype of the conceptus, and not the genotype of the dam, nor heterosis, that determines sensitivity to acetazolamide teratogenesis. They further suggest that this genetic approach may be useful in determining the relative contribution of dam and conceptus for other teratogens.

Investigations of MK-0927 and acetazolamide, both carbonic anhydrase inhibitors (CAIs), showed that urothelial hyperplasia develops in rats and mice, but not in rabbits, dogs, or monkeys. Rats given MK-0927 orally had a rapid onset of the change which regresses often completely despite continued treatment. Increased urinary pH and Na excretion, pharmacologic effects of CAIs, tended to be correlated with lesions. Rats given MK-0927 orally and fed either a 5% potassium phosphate meal or a 5% ammonium chloride meal had reduced urinary pH and/or urinary Na excretion and a reduced incidence of urothelial hyperplasia. Rats given MK-0927 orally and fed a low Na diet had very low urinary Na and essentially no urothelial hyperplasia. It was concluded that a clear relation exists between increased urinary Na excretion and pH, and urothelial hyperplasia induced by CAIs. These results in rats confirm the importance of increased Na and pH as stimuli for the development of urothelial hyperplasia.

Pregnant C57BL/6J mice were treated with 0 or 50 mg of caffeine (CAFF) per kg, and 0, 200 mg/kg (L) or 1,000 mg/kg (H) of acetazolamide (ACZM) during day 9 of gestation (9DPC). Individual fetuses were examined for gross morphological abnormalities and skeletal variations. The increase in fetal malformations seen, especially right forelimb electrodactyly, was augmented at both dose levels of acetazolamide by concomitant exposure to caffeine. Both frequency and severity of ectrodactyly were potentiated by caffeine. Skeletal examination revealed a reduction of the number of ossified cervical and caudal vertebral centra among litters exposed to ACZM at either dose. In either case (ACZM-H, ACZM-L) that effect was augmented by co-administration of CAFF. The first cervical vertebra (C1) appeared to provide the most sensitive index of teratogenic exposure. This study provides evidence that a subteratogenic dose of caffeine can potentiate the teratogenic effect of acetazolamide in C57BL/6J mice when dams are treated on day 9 of gestation. In addition, skeletal examination provided evidence that simultaneous treatment with both agents delayed fetal development. Many litters exposed to ACZM or both agents displayed a reduction in skeletal ossification even in the absence of gross morphological abnormalities, suggesting that ossification can be used as an indicator of prenatal exposure to potentially harmful substances in the C57BL/6 mouse strain.

The WB/ReJ and C57BL/6J strains were compared in their time and dose responses to acetazolamide administered in a single subcutaneous injection regime. WB/ReJ has a genetically determined, high-frequency, transient fetal edema that has maximum expression on day 14 and is resolved by day 18. Acetazolamide, at 1,000 mg/kg, appears to induce edema in WB/ReJ with a time of response on days 9 and 10, and the induced edema follows the same time course of appearance and disappearance as the spontaneous trait. The dose-response analysis is not interpretable in the WB/ReJ and C57BL/6J strains and their reciprocal F1 fetuses because there was significant response only at the highest dose (2,000 mg/kg) used in this study. The time of ectrodactyly response is maximal on day 9 in both WB/ReJ and C57BL/6J strains. The dose-response analysis demonstrates that, for the usual measure of total fetuses with ectrodactyly (or penetrance), the Wb/ReJ and C57BL/6J strains and the WB/ReJ x C57BL/6J F1 (WB.B6F1) have the same slope of the dose-response curve and the strain difference in response can be interpreted as a difference in dosage tolerance. The tolerance of WB/ReJ is twofold greater than that of C57BL/6J. This overdominance of relative resistance to acetazolamide ectrodactyly supports the general finding of directional dominance of relative resistance among genetically different strain pairs. The median effective dose for penetrance of the ectrodactyly response of the reciprocal B6.WBF1 embryo is similar to the WB.B6F1, but the slope of the dose-response curve is significantly different, and a different teratogenic mechanism of response may be involved. Ectrodactyly was predominantly right sided in all genotypes, and, in bilaterally affected fetuses, the right forelimb was more severely affected. An unexpected difference between WB/ReJ and C57BL/6J was found when the laterality of ectrodactyly was analyzed further. There is a significant increase with dosage in bilaterally affected fetuses (a measure of expressivity) in C57BL/6J but not in WB/ReJ, even though the dose-response of total affected fetuses (penetrance) is similar in both strains. In C57BL/6J, the left and right forelimbs are correlated in their responses with the left, requiring approximately a threefold greater dose. The left and right forelimbs are symmetrical in response, and the difference can be interpreted in terms of a developmental (or teratogenic) gradient. In WB/ReJ, the right forelimb has the same dose response as C57BL/6J and requires a twofold greater dose than the right forelimb of C57BL/6J, but the left forelimb has a very flat slope and is not correlated with the response of the right.(ABSTRACT TRUNCATED AT 400 WORDS)

The observations gathered in mouse embryos collected 4, 24 and 48 hours after the administration of a teratogenic doses of acetazolamide to their pregnant mothers strongly suggest that the resulting postaxial defects in the anterior limbs can be the result of a selective perturbation of the inductive process responsible for the genesis of the apical ectodermal ridge, probably secondary to a transient acidosis. The vascular stasis provoked by the treatment provides an explanation for the selective localization of the injury in the forelimb buds and even for its preferential occurrence on the right side.

A continuing survey of the genetic variability of different mouse strains to acetazolamide teratogenesis demonstrated the WB/ReJ strain expresses a high frequency of induced subcutaneous edema in day 15 fetuses. In treated WB/ReJ fetuses, the probability of expression of edema is independent of the expression of forelimb ectrodactyly and, with the dosage regime, there is no significant increase in acetazolamide-induced resorption. It was surprising to find a high frequency of spontaneous edema on day 15 in untreated WB/ReJ fetuses. The spontaneous edema is a transient trait with maximum expression (56%) on day 14, and it is resolved by day 17 without apparent consequence to the survival of previously affected fetuses. There is no sex dimorphism in the liability to express the transient edema. Preliminary genetic crosses to investigate the spontaneous edema were made between WB/ReJ and the C57BL/6J strain, which historically had not be observed to express spontaneous edema. A low frequency of spontaneous edema was observed on day 14 in both C57BL/6J and the reciprocal F1 fetuses. The trait is not additive because there is dominance deviation of the BC1 fetuses in the direction of the F1 fetuses. The data were fitted to a threshold model suggesting that the developmental liability to express the difference in transient edema is determined by more than one gene, but the data can be interpreted by a minimum of two loci with duplicate epistasis. The observed differences in frequencies of edema suggest the genetic model can be tested with relatively few test crosses.

BALB/c female mice were administered several compounds, including 4-ethylsulfonylnaphthalene-1-sulfonamide, acetazolamide, and oxamide, in the diet for six weeks. Fresh urine samples were analyzed three times per week for pH, osmolality, micro-crystals, and protein; and a histopathological evaluation was made of the urothelium at the end of the six weeks test. Incidences of hyperplasia, nodular hyperplasia, vacuolization, ulceration and acute inflammation of the bladder urothelium appeared to be related to the osmolality of the urine and the micro-crystalluria experienced by the mice. Correlation coefficients between lesions and urinary osmolality or crystals were -0.69 (p less than 0.0001) and 0.31 (p less than 0.03), respectively, at the 5% significance level.

In a trial to assess the cognitive impairment attributable to benign acute mountain sickness (AMS) and to acetazolamide, six women and five men, 20-35 years old, ascended from sea-level to 3600 m in 36 h and were assessed for deterioration in performance on psychological tests. Of five sex-matched pairs with a mean age difference of 3.4 years (S.D. +/- 4.4 years), one member took slow-release acetazolamide 500 mg daily and one placebo on a double-blind basis during the ascent and again for an identical course at low altitude 32-38 d later. The unmatched woman took placebo during ascent. Before, during, and after each drug course each subject performed an Environmental Symptom Questionnaire (ESQ) and a psychological test battery consisting of trail-making, paced auditory serial addition test (PASAT), letter-digit code, dual-task cancellation and subtraction, and memory subtests. On ascent, ESQ score deteriorated by an average of 62 points in placebo subjects compared with 32 in acetazolamide subjects (p = 0.055). Deterioration in the psychological test battery was only significant in the PASAT (p less than 0.05) and memory (p less than 0.01) subtests of subjects taking placebo. For those taking acetazolamide, no test showed significant impairment, suggesting it had no detectable cognitive impairment at this dose.

We studied the capacity of various chemicals to promote urinary bladder cancer in male F344 rats after initiation by N-nitroso-n-butyl-(4-hydroxybutyl)amine (BBN). The rats were given initially 0.01% BBN in the drinking-water for 4 wk and then the test compound in the diet for 34 wk. Effects were judged by measuring the formation of preneoplastic lesions papillary or nodular hyperplasia (PN hyperplasia) of the urinary bladder. Administration of 5%, but not 0.5% (w/w) sodium saccharin in the diet significantly increased the incidence and extent of PN hyperplasia. This finding could be related to the induction of cancers in the rat urinary bladder by high levels of saccharin. Sodium ascorbate (5%). DL-tryptophan (5%) and allopurinol (0.02%) also significantly increased the extent of PN hyperplasia in the affected animals, but other test chemicals, such as acetazolamide (0.35%) and quercetin (5%) did not. The results with sodium saccharin and DL-tryptophan were consistent with previous findings and suggest that sodium ascorbate and allopurinol have promoting activities in urinary bladder carcinogenesis in rats. No correlation was found between the extent of crystalluria and promotion of preneoplastic lesions.

The vasoactive agents serotonin, ergotamine, and nicotine potentiate acetazolamide induced forelimb ectrodactyly (missing digits) in Wistar rats. These vasoactive agents administered alone do not produce forelimb ectrodactyly and are not known to be inhibitors of carbonic anhydrase. Additionally, physical clamping of the uterine horns in addition to oral acetazolamide administration increases the frequency of forelimb ectrodactyly, suggesting that decreased uterine blood flow can potentiate acetazolamide teratogenesis. Since the vasoactive agents used in this study are reported to possess uterine vasoconstrictive activity, a decrease in uterine blood flow is a plausible mechanism for the potentiative ability of these agents.

Acetazolamide was administered orally by gavage to pregnant mice twice a day on days six through 15 of gestation at a dose level of 300.0 mg/kg. This treatment produced maternal toxicity as evidenced by significantly reduced food consumption and body weight gain. Embryotoxicity demonstrated by significant reductions in fetal weight and crown-rump length was also observed. Resorption rates were considerably higher (77.9%) for the acetazolamide treated litters when compared to those from the control group (8.23%). A variety of malformations was observed in the acetazolamice-treated rats (rib and vertebral fusion, gastroschisis, tail defects, cleft palate and ectrodactyly). These results further establish acetazolamide as an acceptable positive teratogenic control by demonstration of species and strain susceptibility in the CD-1 mouse.

Acetazolamide, an inhibitor of the enzyme carbonic anhydrase (E.C. 4.2.1.1.), causes a unique congenital anomaly characterized by postaxial reduction of the distal portion of the right forelimb. To gain an understanding of the mechanism of teratogenesis, the activity of carbonic anhydrase in sensitive and resistant mouse strains, and its inhibition by acetazolamide, were examined. Differences in teratologic sensitivity were found not to be attributable to differences in maternal or embryonic drug levels. Enzyme inhibition at acetazolamide concentrations ranging between 10(-11) and 10(-5) M did not differ between the mouse strains when adult erythrocytes or day 12 embryos were assayed. However, in day 10 embryos, the period of maximum teratologic susceptibility, a small strain difference was found which suggested that the form of carbonic anhydrase in the susceptible CBA/J strain at this time is somewhat more sensitive to inhibition by acetazolamide than the form found in the resistant SWV strain. The results suggest further that more than one isozyme of carbonic anhydrase may be present in all three samples.

Acetazolamide and inhibitors of DNA synthesis (hydroxyurea, 5-fluoro-2'-deoxyuridine), RNA synthesis (actinomycin D), and protein synthesis (cycloheximide, emetine) were each administered to pregnant rats together with caffeine at doses where each agent alone caused minimal embryotoxicity. Caffeine co-administered with any of the other agents induced a powerful potentiative response. It is not clear from the present experiments whether much lower caffeine dosage, as normally encountered in humans, would potentiate embryotoxicity due to other agents.

Following exposure to bromodeoxyuridine (BUDR), acetazolamide (ACZM), trypan blue (TRBL), cortisone (CORT), or diphenylhydantoin (DPH), alizarin-stained, cleared fetuses were examined at 18 days postcoitus for unossified cervical vertebral centra; number of ossified caudal vertebrae; number of ribs; and ossification of sternebrae, metatarsals, metacarpals, and phalangeal rows. At all teratogenic doses, in no vehicle-treated groups, and rarely in lower-dose groups, there were significant increases in frequency of unossified cervical centra, the first vertebra (C1) being most often affected, and C7 least often affected. In the high-dose CORT group, there was a significant correlation between unossified C1 and cleft palate. No association between abnormality and reduced ossification of cervical vertebrae was seen in other series examined, nor was there any correlation between litter size and abnormality. With minor complications, the number of ossified caudal vertebrae was significantly reduced after exposure at teratogenic dose levels to all compounds except DPH. Although caudal and cervical ossification were correlated with each other in those series examined, neither was correlated with abnormality. Frequency of 14 ribs was increased in BUDR, ACZM, and TRBL but not CORT or DPH. Other parameters were essentially unaffected. Significantly increased frequency of abnormality, when contrasted with untreated or vehicle-treated groups, was seen at high-dose levels in all but DPH treatments, and mortality was increased in ACZM D9-11, TRBL, and CORT. These studies show that reduced ossification of cervical centra is an excellent indicator of prenatal exposure to noxious substances, and caudal vertebrae appear to be useful as well. Increased frequency of 14 ribs occurred for all strong teratogens utilized if they were administered on day 7 or day 8 postcoitus.

A skeletal variant assay system (SVAS) consisting of a group of 88 spontaneously occurring qualitative variations of the adult mouse skeleton was applied to CD-1 animals that had been exposed in utero to 0, 200, or 1,000 mg/kg/day of the sodium salt of acetazolamide dissolved in distilled water, presented by SC injection of the dam during day 8 or days 9-11 of gestation. Two separate series of experiments were performed, and skeletons were examined at postnatal 62 +/- 2 days. Variation occurred in 62 and 67 characters in the two series. Frequencies of occurrence differed from untreated (UNTD) and vehicle-treated (VEH) values of substantial numbers of variants in a dose related manner for both series in both treatment regimes as did the number of variants which showed significantly different frequencies (P less than .01) in comparisons of experimental with either UNTD or VEH. At the high doses 12 and 16 variants occurred with significantly different frequencies from UNTD in day 8 treatments in the two series, and 15 and 19 variants differed in the days 9-11 treated group. Contrasting high-dose animals with appropriate vehicle controls revealed differences in 13 and 12 variants in day 8 treatment groups and in 18 and 15 variants in days 9-11 groups. Agreement between the two series was good, especially in the D9-11 treatments. Several variants differed significantly from both UNTD and VEH in both series of experiments. Among these were a number which appeared more or less specific to acetazolamide exposure. They include: day 8 treatments--accessory parietal, frontal extension, and 27 presacral vertebrae; day 9-11 treatments--sacral fusions in dorsal processes and vertebral bodies, and caudal fusions and malformations; both sets of treatments--lumbar fusions, and fusions of the transverse processes of the sacral vertebrae. Other importantly affected variants, also seen in exposure to other compounds include: day 8 treatments--abnormal metoptic roots; day 9-11 treatments--accessory mental foramen, foramina transversaria imperfecta of the atlas, arch foramen of the fifth cervical (C) vertebra, malformed sternebrae, fossa olecrani perforata, and fewer than 30 caudal vertebrae; both treatment regimes--parted frontals, accessory transverse foramina in C3-C6, reduced articular processes on the thoracic vertebrae, and 14 ribs. By all criteria applied, the SVAS is able to detect prenatal exposure to acetazolamide in adult skeletons even in the absence of any gross morphological abnormalities.

Acetazolamide, a potent and highly specific inhibitor of carbonic anhydrase, is teratogenic in mammalian embryos and when administered during early limb development causes unique limb defects in a time- and dose-dependent manner. The regenerating urodele limb is often considered to be a good experimental analog of limb development and, if it employs the same mechanisms of tissue interactions during pattern formation, should be susceptible to teratogens which selectively disrupt developmental limb patterning. This study demonstrates that while carbonic anhydrase inhibition is toxic to the red-backed salamander, Plethodon cinereus, it does not have the same teratogenic effect on limb regeneration as seen in mammalian limb development. Several points are considered as to why the regenerating limb, at least in this salamander species, may not be suitable for studying this class of teratogen.

We have demonstrated previously that phenylephrine, a selective postsynaptic alpha-1-adrenergic agonist, significantly potentiates the incidence of acetazolamide-induced right forelimb ectrodactyly in a dose-response manner. As reported herein, phenylephrine also decreases maternal uterine blood flow in a dose-response manner as measured by radioactive microsphere methodology. At the potentiative dose of 12.5 mg/kg phenylephrine decreases uterine blood flow by 86.8% when compared to control. In turn, pretreatment with prazosin, a selective postsynaptic alpha-1-adrenergic antagonist, prevents this large decrease in uterine blood flow and abolishes the potentiation of acetazolamide teratogenesis by phenylephrine. Although the effects of acetazolamide or acetazolamide + phenylephrine on uterine blood flow were not measured the data suggest a correlation between decreased uterine blood flow and potentiation of acetazolamide teratogenesis.