Small Molecule Antipsychotic Aripiprazole Potentiates Ozone-Induced Inflammation in Airway Epithelium.

Inhaled ground level ozone (O3) has well described adverse health effects, which may be augmented in susceptible populations. While conditions, such as pre-existing respiratory disease, have been identified as factors enhancing susceptibility to O3-induced health effects, the potential for chemical interactions in the lung to sensitize populations to pollutant-induced responses has not yet been studied. In the airways, inhaled O3 reacts with lipids, such as cholesterol, to generate reactive and electrophilic oxysterol species, capable of causing cellular dysfunction and inflammation. The enzyme regulating the final step of cholesterol biosynthesis, 7-dehydrocholesterol reductase (DHCR7), converts 7-dehydrocholesterol (7-DHC) to cholesterol. Inhibition of DHCR7 increases the levels of 7-DHC, which is much more susceptible to oxidation than cholesterol. Chemical analysis established the capacity for a variety of small molecule antipsychotic drugs, like Aripiprazole (APZ), to inhibit DHCR7 and elevate circulating 7-DHC. Our results show that APZ and the known DHCR7 inhibitor, AY9944, increase 7-DHC levels in airway epithelial cells and potentiate O3-induced IL-6 and IL-8 expression and cytokine release. Targeted immune-related gene array analysis demonstrates that APZ significantly modified O3-induced expression of 16 genes, causing dysregulation in expression of genes associated with leukocyte recruitment and inflammatory response. Additionally, we find that APZ increases O3-induced IL-6 and IL-8 expression in human nasal epithelial cells from male but not female donors. Overall, the evidence we provide describes a novel molecular mechanism by which chemicals, such as APZ, that perturb cholesterol biosynthesis affect O3-induced biological responses.

Environmental enrichment improves behavioral outcome in the AY-9944 model of childhood atypical absence epilepsy.

Atypical absence seizures are drug resistant in the majority of children with Lennox-Gastaut syndrome and herald a poor neurodevelopmental outcome. Here we studied the effects of environmental enrichment, enriched housing conditions designed to stimulate sensory and motor systems in the brain, on behavioral outcome in mice treated with the cholesterol biosynthesis inhibitor AY-9944 (AY), a clinically relevant model of atypical absence epilepsy. Beginning at postnatal day (P) 2, C3H mice were treated with AY (7.5 mg/kg) every 6 days until P20 and then weaned into enriched or standard cages. After 30 days (∼P50), AY mice from the enriched housing condition exhibited less behavioral hyperactivity and anxiety, improved olfactory recognition, and spatial learning, but no significant reduction in the number of ictal discharges in comparison with their non-enriched cohorts. The beneficial effects of environmental enrichment in AY model were in some behavioral tests gender-specific in favor of males suggesting that other, possibly hormonally mediated mechanisms, may interact with the therapeutic effects of enrichment. Taken together, these data provide a starting point to derive clinical occupational therapies for improving behavioral outcome in cases of intractable childhood seizures.

5-HT2 modulation of AY-9944 induced atypical absence seizures.

We investigated the role of 5-HT(2A) and 5-HT(2C) receptors in atypical absence seizures (AAS) induced by trans-1,4-bis[2-chloro-benzylaminomethyl] cyclohexane, dihydrocholoride (AY-9944). The total duration and number and mean duration of the spontaneous bursts of slow spike-and-wave discharges (SSWD) that characterize the AY model were measured using electrocorticographic (ECoG) recordings in freely moving animals. In a randomized counterbalanced dose response design, rats were treated with either the 5-HT(2A) agonist 1-[2,5-dimethoxy-4-iodophenyl]-2-aminopropane (DOI, 0.5, 1 or 2 mg/kg), the 5-HT(2C) preferring agonist m-chlorophenylpiperazine (mCPP, 1, 2, or 4 mg/kg), the 5-HT(2A) antagonist ketanserin (2.5 or 5 mg/kg), or vehicle. DOI significantly reduced the total duration and number of SSWD. In contrast, mCPP had no effect on total duration or number of SSWD. Ketanserin exacerbated the number of SSWD at 2.5 mg/kg but produced mixed results at 5.0 mg/kg. However, none of the treatments affected the mean SSWD duration. These data support the hypothesis that 5HT(2A) receptors are involved in the pathology of experimental atypical absence seizures.

Partial rescue of retinal function and sterol steady-state in a rat model of Smith-Lemli-Opitz syndrome.

The Smith-Lemli-Opitz syndrome (SLOS) is the first-described in a growing family of hereditary defects in cholesterol biosynthesis, and presents with a spectrum of serious abnormalities, including multiple dysmorphologies, failure to thrive, cognitive and behavioral impairments, and retinopathy. Using a pharmacologically induced rat model of SLOS that exhibits key hallmarks of the disease, including progressive retinal degeneration and dysfunction, we show that a high-cholesterol diet can substantially correct abnormalities in retinal sterol composition, with concomitant improvement of visual function, particularly within the cone pathway. Although histologic degeneration still occurred, a high-cholesterol diet reduced the number of pyknotic photoreceptor nuclei, relative to animals on a cholesterol-free diet. These findings demonstrate that cholesterol readily crosses the blood-retina barrier (unlike the blood-brain barrier) and suggest that cholesterol supplementation may be efficacious in treating SLOS-associated retinopathy.

GABAB receptor antagonism abolishes the learning impairments in rats with chronic atypical absence seizures.

Chronic atypical absence seizures are a component of the Lennox-Gastaut syndrome, a disorder invariably associated with severe cognitive impairment in children. However, the cause of this intellectual delay remains unclear. The AY9944 model of chronic atypical absence seizures in rats reliably reproduces the electrographic, behavioral, pharmacological and cognitive features of clinical atypical absence. Using this model, we tested the hypothesis that the cognitive impairment associated with this disorder involves a gamma-aminobutyric acid B (GABA(B)) receptor-mediated mechanism. Therefore, we examined the effect of a specific, high affinity GABA(B) receptor antagonist, CGP35348, on the atypical absence seizures, the working memory deficits, and the altered long-term potentiation that we have observed in the AY9944 model. CGP35348 blocked atypical absence seizures, restored long-term potentiation to normal level, and reversed the cognitive deficit in the AY9944-treated animals. However, dose-response studies showed that lower doses of CGP35348 that failed to influence atypical absence seizure activity, completely reversed the spatial working memory deficit. These data suggest that GABA(B) receptor-mediated mechanisms are responsible for the cognitive dysfunction in the AY9944 model of chronic atypical absence seizures and further, that their cognitive impairment is independent of the seizure activity. The data raise the possibility that GABA(B) receptor antagonists may have therapeutic potential for the treatment of cognitive impairment in epilepsy syndromes where atypical absence seizures are a component.

Retinal degeneration in a rodent model of Smith-Lemli-Opitz syndrome: electrophysiologic, biochemical, and morphologic features.

OBJECTIVE: To assess the electrophysiologic, histologic, and biochemical features of an animal model of Smith-Lemli-Opitz syndrome (SLOS). METHODS: Sprague-Dawley rats were treated with AY9944, a selective inhibitor of 3beta-hydroxysterol-Delta(7)-reductase (the affected enzyme in SLOS). Dark- and light-adapted electroretinograms were obtained from treated and control animals. From each animal, 1 retina was analyzed by microscopy, and the contralateral retina plus serum samples were analyzed for sterol composition. The main outcome measures were rod and cone electroretinographic amplitudes and implicit times, outer nuclear layer (ONL) thickness, rod outer segment length, pyknotic ONL nucleus counts, and the 7-dehydrocholesterol/cholesterol mole ratio in the retina and serum. RESULTS: By 10 weeks' postnatal age, rod and cone electroretinographic wave amplitudes in AY9944-treated animals were significantly reduced and implicit times were significantly increased relative to controls. Maximal rod photoresponse and gain values were reduced approximately 2-fold in treated animals relative to controls. The ONL thickness and average rod outer segment length were reduced by approximately 18% and 33%, respectively, and ONL pyknotic nucleus counts were approximately 4.5-fold greater in treated animals relative to controls. The retinal pigment epithelium of treated animals contained massive amounts of membranous/lipid inclusions not routinely observed in controls. The 7-dehydrocholesterol/cholesterol mole ratios in treated retinas and serum samples were approximately 5:1 and 9:1, respectively, whereas the ratios in control tissues were essentially zero. CONCLUSIONS: This rodent model exhibits the key biochemical hallmarks associated with SLOS and displays electrophysiologic deficits comparable to or greater than those observed in the human disease. Clinical Relevance These results predict retinal degeneration in patients with SLOS, particularly those with the more severe (type II) form of the disease, and may be more broadly relevant to other inborn errors of cholesterol biosynthesis. This animal model may also be of use in evaluating therapeutic treatments for SLOS and in understanding the slow phototransduction kinetics observed in patients with SLOS.

Hormonal regulation of atypical absence seizures.

A time course study that examined the effects of the female estrous cycle on the chronic slow spike-and-wave discharges (SSWDs), gamma-aminobutyric B receptor (GABA(B)R) binding, and GABA(B)R protein expression was conducted in Long Evans hooded rats treated during development with a cholesterol synthesis inhibitor AY9944 (AY). In addition, a pharmacological study using the hormones progesterone, 17 beta-estradiol, mifepristone (intracellular progesterone receptor antagonist), tamoxifen (intracellular estrogen receptor antagonist), and allopregnanolone (progesterone metabolite) was performed to determine their effects on AY-induced seizures. The data indicate that there is a significant increase in both the duration of SSWD and GABA(B)R binding in the AY model, during the proestrus stage of the estrous cycle, the stage during which the levels of progesterone are at their highest. No changes in GABA(B)R1a or R2 protein levels were observed. In addition, the administration of both progesterone and allopregnanolone exacerbated seizures in the AY model, whereas 17 beta-estradiol attenuated the SSWD duration. Neither mifepristone nor tamoxifen blocked the effects of progesterone and 17 beta-estradiol, respectively, on SSWD duration in the AY model, suggesting that these two sex hormones are working in a manner independent of their intracellular receptors. These data suggest an important role for steroid hormones in the regulation and maintenance of AY-induced atypical absence seizures.

Role of cholesterol in embryonic development.

We showed previously that 3 distal inhibitors of cholesterol synthesis are highly teratogenic in rats. AY 9944 and BM 15766 inhibit 7-dehydrocholesterol reductase, which catalyzes the last step of cholesterol synthesis, and triparanol inhibits Delta(24)-dehydrocholesterol reductase, which catalyzes the last step in another pathway. These molecules cause holoprosencephalic brain anomalies. Under certain experimental conditions, other anomalies (of the limbs and male genitalia) are also observed. Assays performed by gas chromatography-mass spectrometry (GC-MS) show hypocholesterolemia and an accumulation of precursors. These data indicate that this animal model can be considered a model of Smith-Lemli-Opitz syndrome. Smith-Lemli-Opitz syndrome is a recessive autosomal genetic disease characterized by malformations (microcephaly, corpus callosum agenesis, holoprosencephaly, and mental retardation), male pseudohermaphroditism, finger anomalies, and failure to thrive. The syndrome has been attributed to a deficit in 7-dehydrocholesterol reductase. As assayed by GC-MS, the sterol status of these patients indicates severe hypocholesterolemia and an accumulation of precursors: 7-dehydrocholesterol, 8-dehydrocholesterol, and oxidized derivatives. The presence of 7-dehydrocholesterol in the serum of patients is pathognomonic of the disease. The developmental gene Shh (sonic hedgehog) plays a key role in brain, limb, and genital development; it was shown recently that the Shh protein has to be covalently linked to cholesterol to be active. This is the first time that a posttranslational function has been attributed to cholesterol. There is an obvious relation between Shh dysfunction and the malformations observed in our experiments and in patients with Smith-Lemli-Opitz syndrome. However, the exact relation remains to be clarified. It is clear, however, that the role of cholesterol in embryonic development must be taken into account.

Absence of ventral cell populations in the developing brain in a rat model of the Smith-Lemli-Opitz syndrome.

The Smith-Lemli-Opitz syndrome (SLOS) is an autosomal recessive condition involving craniofacial and central nervous system malformations with occasional holoprosencephaly (HPE). It is caused by a defect in the 7-dehydrocholesterol (7-DHC) reductase, the enzyme catalyzing the last step of cholesterol biosynthesis. Treatment of pregnant rats with inhibitors of 7-DHC reductase, either AY9944 or BM15.766, has provided a valuable model to study the pathogenesis in SLOS. Recently, cholesterol has been shown to be involved in the post-translational activation of the signaling protein Sonic Hedgehog. To identify the early defects associated with HPE in a rat model of SLOS, and to compare the phenotype of the treated embryos with that of the Shh(-/-) mutants, we examined brain morphology and expression of three developmental genes (Shh, Otx2, and Pax6 ) in 23-somite stage embryos from AY9944-treated dams. We report clearly abnormal morphology of the developing brain, concerning primarily the ventral aspect of the neural tube. We observed a reduced or absent expression of Shh and Otx2 in their ventral domain associated with extended ventral expression of Pax6. The results suggest an absence of the midline ventral cell type at all levels of the cranial neural tube. They provide further evidence that cholesterol-deficiency-induced HPE originates from impaired Shh signaling activity in the ventral neural tube.

Abnormal cholesterol biosynthesis as in Smith-Lemli-Opitz syndrome disrupts normal skeletal development in the rat.

Smith-Lemli-Opitz syndrome (SLOS) in human infants is a common autosomal recessive malformation syndrome (estimated incidence, 1:20,000). It is characterized clinically by congenital anomalies, especially craniofacial and limb defects, and biochemically by a defect in 7-dehydrocholesterol-delta7-reductase activity (7DHC-reductase), the final enzyme in cholesterol biosynthesis. In previous studies, early administration of the 7DHC-reductase inhibitor AY9944 to pregnant rats resulted in a high frequency of holoprosencephaly, relevant to craniofacial anomalies of SLOS. In order to test the effect of AY9944 on limb development, we treated dams on gestation day 7 (GD7), which delays the biochemical defect to about GD13 to GD14. Sera were sampled on GD12, GD14, and GD21 and cholesterol and dehydrocholesterols (7DHC and 8DHC) were measured by gas-chromatography-mass spectrometry (GC-MS), as for the diagnosis of SLOS. GD21 fetuses were examined for gross malformations and skeletal development. In treated dams, the SLOS biochemical marker 7DHC accounted for one fourth and one third of total sterols, respectively, on GD12 and GD14, and cholesterolemia on these two gestation days was reduced by 50% and 43%, respectively, as compared with control values. This maternal metabolic defect was associated with decrease in fetal weight and delayed ossification. In addition, scapular malformations were observed in four fetuses from three litters. The malformations could have been caused by the same mechanism as holoprosencephaly after early treatment with AY9944. These cholesterol-deficiency-based malformations could have a common cause in the abnormal expression of Hedgehog or other developmental gene proteins, and may thus explain various congenital polymalformative syndromes in humans, including SLOS.

Pharmacological models of generalized absence seizures in rodents.

A number of animal models of generalized absence seizures in rodents are described. These include absence seizures induced by gamma-hydroxybutyrate (GHB), low dose pentylenetetrazole, penicillin, THIP, and AY-9944. All of these models share behavioral and EEG similarity to human absence seizures and show pharmacologic specificity for antiabsence drugs such as ethosuximide and trimethadione. Moreover, the absence seizures induced by these agents are exacerbated by GABAergic agonists, a property unique to experimental absence seizures. These models are predictable, reproducible, and easy to standardize. They are useful both in studying mechanisms of pathogenesis of absence seizures as well as in screening for antiabsence activity of potential antiepileptic drugs.

Teratogenic effect of the cholesterol synthesis inhibitor AY 9944 on rat embryos in vitro.

AY 9944 [trans-1,4-bis(2-chlorobenzylaminomethyl) cyclohexane dihydrochloride] is an amphiphilic cationic molecule. This chemical is an established inhibitor of cholesterol synthesis and is teratogenic in rats. The mechanisms of this teratogenicity remain to be clarified. This study used cultured rat whole embryos to ascertain whether AY 9944 had a direct effect on embryos, or whether its action was indirect, via the maternal cholesterol metabolism. Four experimental conditions were investigated: (A) controls; (B) 10 day untreated embryos were cultured in serum of treated rats; (C) 10 day untreated embryos were cultured in serum containing added AY 9944 (0-1,000 micrograms/ml); and (D) 10 day embryos from females treated on day 4 of gestation were cultured in normal serum. In group B there was no growth retardation; some slight nonspecific abnormalities were not significant. In group C, direct addition of AY 9944 to culture medium retarded growth and differentiation in a dose-dependent manner. No malformation was observed, but histological examinations showed numerous areas of cell necrosis, especially in the CNS. In group D, not only was growth retardation observed, but also characteristic malformations of AY 9944 teratogenesis, including pituitary agenesis. These results show that AY 9944 teratogenicity is initiated prior to day 10.

Cholesterol prevents the teratogenic action of AY 9944: importance of the timing of cholesterol supplementation to rats.

These studies were conducted to determine whether dietary cholesterol supplementation could prevent fetal malformations induced by the amphipathic drug AY 9944, which is well known as a cholesterol biosynthesis inhibitor, and to investigate whether the plasma maternal sterol level and the nature of the sterols found in treated Wistar rats could explain this prevention. Pituitary agenesis was the most constant element of holoprosencephaly when AY 9944 was administered on d 4 of gestation at two dosages, 50 or 75 mg/kg. The rate of malformed fetuses was dose related. A strong negative correlation was established between maternal plasma sterol levels on d 10 of gestation (day of pituitary gland formation) and the rate of fetal anomalies (r = -0.97, P less than 0.01). Supplementation of AY 9944-treated rats with cholesterol had an obvious preventive action on fetal malformations. When cholesterol was added to the diet the same day as AY 9944 treatment and maintained until d 15, the prevention of malformations was almost complete. When the supplementation was initiated later, the prevention of anomalies decreased. The nature of plasma maternal sterols shows that the cholesterol supplementation modifies significantly the ratio of cholesterol to 7-dehydrocholesterol in treated rats. Therefore, maternal plasma sterol perturbations may play a role in the teratogenic action of AY 9944.

Newborn response to cationic amphiphilic drugs.

Administration of various cationic amphiphilic drugs in utero results in induction of a phospholipid storage disorder in many tissues, particularly in lungs. In addition to the phospholipidosis in utero, drug exposure results in toxicity to the offspring; newborn rats die within 48 h of birth. Although drug-induced pulmonary pathological changes appear to be involved in the observed mortality, this relationship remains unclear. In contrast to mammals, administration of cationic amphiphilic drugs to the chick embryo seems not to induce phospholipid storage in the tissues examined. Treatment of newborn rats directly with these drugs also induces phospholipidosis in several tissues including lung and kidney; however, mortality does not occur. Concurrent administration of phenobarbital and chlorphentermine reduces or prevents amphiphilic drug-induced phospholipid storage in newborn rat lung and kidney. Modification of chlorphentermine actions by phenobarbital may be caused by alterations in amphiphilic drug excretion, metabolism, and catabolic phospholipase activity. Evidence thus indicates that regardless of age, animals appear susceptible to the effects of cationic amphiphilic drugs; however, species and tissues examined, as well as specific drug administration, play an important role in the observed qualitative and quantitative responses.

Teratogenic action and embryo lethality of AY 9944R. Prevention by a hypercholesterolemia-provoking diet.

Embryomortality and teratogenesis provoked by inhibitors of cholesterol synthesis are well demonstrated. Teratogenic action is particularly reflected by holoprosencephalies, but also by uro-genital abnormalities. A hypercholesterolemia-provoking diet has been shown to be completely effective for preventing holoprosencephaly, but only partially so for preventing the uro-genital malformations and fetal mortality. It is thus possible that the two types of abnormalities are governed by different mechanisms. In addition, the diet itself, whose hypercholesterolemic effect is considerable, has certain disadvantages. It seems to have a certain effect on fetal mortality and could be responsible for several uro-genital malformations. This deserves further study.

Interpretation of isolated agenesis of the pituitary.

Cholesterol synthesis inhibitors administered to rats caused more or less complete forms of the holoprosencephalic syndrome, consisting of severe abnormalities of the brain, sense organs and pituitary. The absence of the pituitary was also observed in fetuses without externally visible cephalic abnormalities. These observations suggest that the isolated absence of the pituitary is the lesser form of the holoprosencephalic syndrome. This interpretation is also valid for cases of isolated absence of the pituitary observed in humans.

[Teratogenic action of an inhibitor of cholesterol synthesis in Wistar and Sprague-Dawley rats]. / Action tératogène d'un inhibiteur de la synthèse du cholestérol chez les rats Wistar et Sprague-Dawley.

Sprague-Dawley rats are sensitive to the teratogenic action of AY 9944, an inhibitor of cholesterol synthesis, but the dose of inhibitor necessary to induce the same rate of characteristic malformations is twice as large for Sprague-Dawley as for Wistar rats. This variation is probably related to differences in levels of blood cholesterol in the strains and demonstrates a relationship between teratogenicity and metabolic disturbances.