Effect of cytochrome P450 3A4 on tissue distribution of humantenmine, koumine, and gelsemine, three alkaloids from the toxic plant Gelsemium.

Humantenmine, koumine, and gelsemine are three indole alkaloids found in the highly toxic plant Gelsemium. Humantenmine was the most toxic, followed by gelsemine and koumine. The aim of this study was to investigate and analyze the effects of these three substances on tissue distribution and toxicity in mice pretreated with the Cytochrome P450 3A4 (CYP3A4) inducer ketoconazole and the inhibitor rifampicin. The in vivo test results showed that the three alkaloids were absorbed rapidly and had the ability to penetrate the blood-brain barrier. At 5 min after intraperitoneal injection, the three alkaloids were widely distributed in various tissues and organs, the spleen and pancreas were the most distributed, and the content of all tissues decreased significantly at 20 min. Induction or inhibition of CYP3A4 in vivo can regulate the distribution and elimination effects of the three alkaloids in various tissues and organs. Additionally, induction of CYP3A4 can reduce the toxicity of humantenmine, and vice versa. Changes in CYP3A4 levels may account for the difference in toxicity of humantenmine. These findings provide a reliable and detailed dataset for drug interactions, tissue distribution, and toxicity studies of Gelsemium alkaloids.

Toxicological effects caused by environmental relevant concentrations of ketoconazole in Chironomus sancticaroli (Diptera, Chironomidae) larvae evaluated by oxidative stress biomarkers.

Ketoconazole (KTZ), a broad-spectrum fungicidal drug, has been a significant problem in recent decades due to its toxic action on non-target aquatic organisms. Thus, the present study aimed to evaluate determine the effects that environmental relevant concentration of the commercial formulation of KTZ can exert on benthic macroinvertebrates, more specifically on larvae of the insect Chironomus sancticaroli. Acute toxicity tests with KTZ indicated lethal concentration (LC50) of 9.9 µg/L. Analyses of prolonged exposure to KTZ (chronic toxicity) indicated an increase in the rate of mentum deformity by approximately 3 times at concentrations of 0.6 and 2.4 µg/L. All biomarkers analyzed showed an increase after exposure to KTZ (0.6 and 2.4 µg/L), with average values of 115 % for superoxide dismutase (SOD), 63 % for catalase (CAT), 111 % for glutathione S-transferase (GST) and 59 % for malonaldehyde (MDA) in C. sancticaroli larvae. Thus, the toxic effects on survival, development (length and weight), mentum and redox responses caused by commercial KTZ in low concentrations were observed on C. sancticaroli larvae. In addition, the results suggest that biochemical biomarkers can be used for studies involving environmental disturbances.

Chemicals of environmental concern as inhibitors of human placental 3ß-hydroxysteroid dehydrogenase 1 and aromatase: Screening and docking analysis.

Many environmental pollutants act as endocrine-disrupting compounds by inhibiting human placental 3ß-hydroxysteroid dehydrogenase/Δ5-4 isomerase type 1 (HSD3B1) and aromatase (CYP19A1) activities. In this study, we screened 13 chemicals of environmental concern for their ability to inhibit human HSD3B1 and CYP19A1 by measuring the conversion of pregnenolone to progesterone for HSD3B1 activity and the conversion of testosterone to 17ß-estradiol for CYP19A1 activity in human JEG-3 choriocarcinoma cell microsomes. HSD3B1 had an apparent Km of 0.323 µM and an apparent Vmax of 0.111 nmol/mg/min and CYP19A1 had an apparent Km of 56 nM and an apparent Vmax of 0.177 nmol/mg protein/min. 17ß-Estradiol, bisphenol A, and bisphenol AF competitively inhibited HSD3B1 with Ki values of 0.8, 284.1, and 141.2 µM, respectively, while diethylstilbestrol had a mixed inhibition on human HSD3B1 with the Ki of 8.0 µM. Ketoconazole, bisphenol A, and bisphenol AF noncompetitively inhibited CYP19A1 with Ki values of 10.3, 54.4, and 45.7 µM, respectively, while diethylstilbestrol and zearalenone competitively suppressed CYP19A1 with Ki values of 63.0 and 16.6 µM, respectively. Docking analysis showed that 17ß-estradiol, diethylstilbestrol, bisphenol A, and bisphenol AF bound the steroid binding pocket facing the catalytic residues Y155 and K159 of HSD3B1, and that ketoconazole, bisphenol A, and bisphenol AF bound heme binding pocket while diethylstilbestrol and zearalenone bound the steroid binding site of CYP19A1. In conclusion, 17ß-estradiol, diethylstilbestrol, bisphenol A, and bisphenol AF are human HSD3B1 inhibitors, and ketoconazole, zearalenone, diethylstilbestrol, bisphenol A, and bisphenol AF are human CYP19A1 inhibitors.

Arylacetamide deacetylase knockout mice are sensitive to ketoconazole-induced hepatotoxicity and adrenal insufficiency.

Orally administered ketoconazole may rarely induce liver injury and adrenal insufficiency. A metabolite formed by arylacetamide deacetylase (AADAC)-mediated hydrolysis has been observed in cellulo studies, and it is relevant to ketoconazole-induced cytotoxicity. This study tried to examine the significance of AADAC in ketoconazole-induced toxicity in vivo using Aadac knockout mice. Oral administration of 150 mg/kg ketoconazole resulted in the area under the plasma concentration-time curve values of ketoconazole and N-deacetylketoconazole, a hydrolyzed metabolite of ketoconazole, in Aadac knockout mice being significantly higher and lower than those in wild-type mice, respectively. With the administration of ketoconazole (300 mg/kg/day) for 7 days, Aadac knockout mice showed higher mortality (100%) than wild-type mice (42.9%), and they also showed significantly higher plasma alanine transaminase and lower corticosterone levels, thus representing liver injury and steroidogenesis inhibition, respectively. It was suggested that a higher plasma ketoconazole concentration likely accounts for the inhibition of the synthesis of corticosterone, which has anti-inflammatory effects, in the adrenal gland in Aadac KO mice. In Aadac knockout mice, hepatic mRNA levels of immune- and inflammation-related factors were increased by the administration of 300 mg/kg ketoconazole, and the increase was restored by the replenishment of corticosterone (40 mg/kg, s.c.) along with recoveries of plasma alanine transaminase levels. In conclusion, Aadac defects exacerbate ketoconazole-induced liver injury by inhibiting glucocorticoid synthesis and enhancing the inflammatory response. This in vivo study revealed that the hydrolysis of ketoconazole by AADAC can mitigate ketoconazole-induced toxicities.

Effects of nanoplastic on toxicity of azole fungicides (ketoconazole and fluconazole) in zebrafish embryos.

The ubiquity of nanoplastics (NPs) raises concerns about their interactions and combined toxicity with other common contaminants. Although azoles are present throughout the natural environment, their interactions with NP are not well known. We investigated the effects of polystyrene (PS) NP on the toxicity of ketoconazole (KCZ) and fluconazole (FCZ) in zebrafish embryos using the developmental toxicity, oxidative-stress-related biochemical parameters, and expression of genes related to neurotoxicity (ache), cardiotoxicity (gata4, bmp4), inflammation (il1b), oxidative stress (sod1, sod2, cyp1a), and apoptosis (bax, bcl2). Co-exposure to NP (1 mg/L) and KCZ/FCZ (1 mg/L) for 96 h reduced the hatching rate, survival rate, and heart rate and increased the malformation rate and catalase activity. The bax/bcl2 ratio, an apoptosis indicator, was higher after NP, KCZ, or FCZ treatment. However, the bax/bcl2 ratio after exposure to NP + KCZ or NP + FCZ was much higher than that after single exposure. Overall, the results indicated that NP aggravated the toxicity of azole by significantly increasing the reactive oxygen species, lipid peroxidation and altering the expression of oxidative-stress- and apoptosis-related genes. The interactive toxicity of PS NP with KCZ/FCZ reported in this study emphasises the need for caution in the release of azole fungicides in the environment.

Studying the ophthalmic toxicity potential of developed ketoconazole loaded nanoemulsion in situ gel formulation for ophthalmic administration.

Ocular fungal infections are one of the essential reasons for vision loss, especially in developing countries for tropical regions. Ketoconazole (KZ), a broad-spectrum antifungal drug, is a lipophilic compound and practically insoluble in water. Since topical ophthalmic drug delivery confronts low bioavailability, an in situ gel formulation is designed to improve the residence time and consequently the bioavailability. Safety of the developed formulation as a carrier for ophthalmic drug delivery was measured using three different methods: MTT assay for measuring cell viability in which the human retinal pigmentation epithelial cells (RPE) were used, HET-CAM as a borderline method between in vivo and in vitro techniques for investigating the irritation potential of the chosen formulation which was done by adding formulation directly on the CAM surface and visually monitoring the vessels in terms of irritation reactions, and finally the modified Draize test for evaluating tolerability of the selected formulation on eyes. According to our results from the MTT test, cell viability for KZ-NE in situ gel formulation at 0.1% concentration was acceptable. The results obtained from the HET-CAM investigation didn't show any sign of vessel injury on the CAM surface for prepared formulation. Additionally, during 24 hours, the developed formulation was tolerable by rabbit eyes. Regarding our results, KZ-NE in situ gel formulation was non-irritant and non-toxic and can be well-tolerated and presented as an applicable vehicle for ophthalmic delivery of the anti-fungal drug.

Ecotoxicological effect of ketoconazole on the antioxidant system of Daphnia similis.

The occurrence of emerging pharmaceutical pollutants (i.e. small drugs, antibiotics) present in aquatic environments shown to be a current environmental problem still without apparent solution. In this regard, the use of ecotoxicological techniques has been shown fundamental for the appraisal of damage to affected living organisms. Herein, ecotoxicological tests were conducted, focusing on the evaluation of the effects of ketoconazole (KTZ) on the antioxidant system of the model body Daphnia similis. In order to study the biochemical changes caused by KTZ in the antioxidant system, the enzymatic biomarkers glutathione S-transferase (GST), catalase (CAT), and ascorbate peroxidase (APX) were monitored. Toxicological tests were conducted using KTZ concentrations (0-10 µg·L-1). Prolonged exposure to KTZ (336 h) caused changes upon the expression of antioxidant enzymes and simultaneously affected the reproductive system in those organisms. Moreover, a decrease in GST and APX activity was observed caused by KTZ exposure, respectively 79.2% (3.53 µmol min-1 mg-1 protein) and 24.4% (0.88 µmol min-1 mg-1 protein). On the other hand, it was observed an increase of 27% (0.17 µmol min-1 mg-1 protein) in CAT activity. Through this study, it was possible to observe the toxicological effects of KTZ, which proves its action as an oxidative stress-inducing agent and endocrine modifier in daphnids organisms.

Classical toxicity endpoints in female rats are insensitive to the human endocrine disruptors diethylstilbestrol and ketoconazole.

Developmental exposure to endocrine disrupting chemicals can have negative consequences for reproductive health in both men and women. Our knowledge about how chemicals can cause adverse health outcomes in females is, however, poorer than our knowledge in males. This is possibly due to lack of sensitive endpoints to evaluate endocrine disruption potential in toxicity studies. To address this shortcoming we carried out rat studies with two well-known human endocrine disruptors, diethylstilbestrol (DES) and ketoconazole (KTZ), and evaluated the sensitivity of a series of endocrine related endpoints. Sprague-Dawley rats were exposed orally from gestational day 7 until postnatal day 22. In a range-finding study, disruption of pregnancy-related endpoints was seen from 0.014 mg/kg bw/day for DES and 14 mg/kg bw/day for KTZ, so doses were adjusted to 0.003; 0.006; and 0.0012 mg/kg bw/day DES and 3; 6; or 12 mg/kg bw/day KTZ in the main study. We observed endocrine disrupting effects on sensitive endpoints in male offspring: both DES and KTZ shortened anogenital distance and increased nipple retention. In female offspring, 0.0012 mg/kg bw/day DES caused slightly longer anogenital distance. We did not see effects on puberty onset when comparing average day of vaginal opening; however, we saw a subtle delay after exposure to both chemicals using a time-curve analysis. No effects on estrous cycle were registered. Our study shows a need for more sensitive test methods to protect the reproductive health of girls and women from harmful chemicals.

Identification of Translational microRNA Biomarker Candidates for Ketoconazole-Induced Liver Injury Using Next-Generation Sequencing.

Drug-induced liver injury (DILI) is a leading cause of acute liver failure. Reliable and translational biomarkers are needed for early detection of DILI. microRNAs (miRNAs) have received wide attention as a novel class of potential DILI biomarkers. However, it is unclear how DILI drugs other than acetaminophen may influence miRNA expression or which miRNAs could serve as useful biomarkers in humans. We selected ketoconazole (KCZ), a classic hepatotoxin, to study miRNA biomarkers for DILI as a proof of concept for a workflow that integrated in vivo, in vitro, and bioinformatics analyses. We examined hepatic miRNA expression in KCZ-treated rats at multiple doses and durations using miRNA-sequencing and correlated our results with conventional DILI biomarkers such as liver histology. Significant dysregulation of rno-miR-34a-5p, rno-miR-331-3p, rno-miR-15b-3p, and rno-miR-676 was associated with cytoplasmic vacuolization, a phenotype in rat livers with KCZ-induced injury, which preceded the elevation of serum liver transaminases (ALT and AST). Between rats and humans, miR-34a-5p, miR-331-3p, and miR-15b-3p were evolutionarily conserved with identical sequences, whereas miR-676 showed 73% sequence similarity. Using quantitative PCR, we found that the levels of hsa-miR-34a-5p, hsa-miR-331-3p, and hsa-miR-15b-3p were significantly elevated in the culture media of HepaRG cells treated with 100 µM KCZ (a concentration that induced cytotoxicity). Additionally, we computationally characterized the miRNA candidates for their gene targeting, target functions, and miRNA/target evolutionary conservation. In conclusion, we identified miR-34a-5p, miR-331-3p, and miR-15b-3p as translational biomarker candidates for early detection of KCZ-induced liver injury with a workflow applicable to computational toxicology studies.

Melatonin ameliorates some biochemical alterations following ketoconazole administration in rats.

Background Ketoconazole (Keto), an antifungal drug and a common therapeutic option in the treatment of advanced prostate cancer, is known to cause reproductive dysfunctions. Like Keto, melatonin has antifungal and anticarcinogenic actions. Moreover, the hormone has been used to reverse the damaging effects of different toxicants on the reproductive system. Therefore, this study investigated the effects of Keto with/without melatonin on selected biomarkers in rats. Methods Forty rats of 10 animals per group were used in this study, which lasted for 6 weeks. The control group was administered with saline (0.1 mL/day), while group 2 was administered with Keto during the last 3 weeks of experiment; however, in groups 3 and 4, Keto was administered during the first 3 weeks; thereafter, they were administered with saline and melatonin, respectively, during the subsequent 3 weeks. Keto and melatonin were administered at 100 and 10 mg/kg b.w./day (p.o.), respectively. Results The central effects of Keto are independent of the follicle stimulating hormone (FSH) and prolactin; however, relative to the control group, the drug significantly decreased the gonadotrophin releasing hormone (GNRH) and the luteinizing hormone (LH), substantiated by the corresponding significant decreases in sperm count and sperm morphology. Keto caused significant elevations in malondialdehyde (MDA) and lactate dehydrogenase (LDH) and a significant decrease in catalase (CAT) compared with the control group. Moreover, the drug triggered pro-inflammatory events. In group 3 (Keto recovery), MDA and uric acid levels were returned to the baseline (i.e. control), but not GNRH, LH, C-reactive protein (CRP), LDH, and CAT. Treatment with melatonin after Keto administration caused significant increases in FSH, LH, superoxide dismutase, total antioxidant capacity (TAC), sperm count, and sperm morphology but significant decreases in MDA and CRP, relative to groups 2 and 3. Conclusions Melatonin ameliorates some biochemical alterations following ketoconazole administration.

New diphenylphosphane derivatives of ketoconazole are promising antifungal agents.

Four new derivatives of ketoconazole (Ke) were synthesized: diphenylphosphane (KeP), and phosphane chalcogenides: oxide (KeOP), sulphide (KeSP) and selenide (KeSeP). These compounds proved to be promising antifungal compounds towards Saccharomyces cerevisiae and Candida albicans, especially in synergy with fluconazole. Simulations of docking to the cytochrome P450 14α-demethylase (azoles' primary molecular target) proved that the new Ke derivatives are capable of inhibiting this enzyme by binding to the active site. Cytotoxicity towards hACSs (human adipose-derived stromal cells) of the individual compounds was studied and the IC50 values were higher than the MIC50 for C. albicans and S. cerevisiae. KeP and KeOP increased the level of the p21 gene transcript but did not change the level of p53 gene transcript, a major regulator of apoptosis, and decreased the mitochondrial membrane potential. Taken together, the results advocate that the new ketoconazole derivatives have a similar mechanism of action and block the lanosterol 14α-demethylase and thus inhibit the production of ergosterol in C. albicans membranes.

Implementation of a functional endpoint to the zebrafish embryotoxicity test to evaluate craniofacial abnormalities.

The inclusion of a read-out to detect functional consequences of craniofacial alterations in the zebrafish embryotoxicity test will allow to evaluate these alterations which are difficult to assess morphologically, and to detect alterations in cranial nerves functions leading to impairment of jaw movements. In this study we have established an ingestion test in zebrafish larvae younger than 120 hpf. To overcome the challenge of evaluating larvae which still do not present independent feeding behaviour, we have tested the ability of 72, 96 or 102 hpf larvae to ingest food mixed with fluorescent microspheres under several conditions (dark/light, with/without shaking) to find the best experimental set-up for the test. We have included the investigation of two substances as potential positive controls: ketoconazole and tricaine. Ketoconazole 10 µM exposure during development produced significant embryotoxic effects including a characteristic craniofacial alteration pattern consisting in impaired development of brain, nasal cavity, mouth opening and jaw, as well as a significant decrease in food intake. Tricaine exposure at 380 µM during the food availability period significantly decreased the food intake. The method proposed will be a useful alternative tool to animal testing to detect compounds inducing adverse effects on craniofacial development.

Physiological and drug-induced changes in blood levels of adrenal steroids and their precursors in cynomolgus monkeys: An application of steroid profiling by LC-MS/MS for evaluation of the adrenal toxicity.

The adrenal gland is the most common toxicological target of drugs within the endocrine system, and inhibition of adrenal steroidogenesis can be fatal in humans. However, methods to evaluate the adrenal toxicity are limited. The aim of the present study was to verify the usefulness of simultaneous measurement of blood levels of multiple adrenal steroids, including precursors, as a method to evaluate drug effects on adrenal steroidogenesis in cynomolgus monkeys. With this aim, physiological and drug-induced changes in blood levels of adrenal steroids, including cortisol, aldosterone, androgen, and their precursors were examined. First, for physiological changes, intraday and interday changes in blood steroid levels were examined in male and female cynomolgus monkeys. The animals showed circadian changes in steroid levels that are similar to those in humans, while interday changes were relatively small in males. Next, using males, changes in blood steroid levels induced by ketoconazole and metyrapone were examined, which suppress adrenal steroidogenesis via inhibition of CYP enzymes. Consistent with rats and humans, both ketoconazole and metyrapone increased the deoxycorticosterone and deoxycortisol levels, probably via CYP11B1 inhibition, and the increase was observed earlier and with greater dynamic range than the changes in cortisol level. Changes in other steroid levels reflecting the drug mechanisms were also observed. In conclusion, this study showed that in cynomolgus monkeys, simultaneous measurement of blood levels of adrenal steroids, including precursors, can be a valuable method to sensitively evaluate drug effects on adrenal steroidogenesis and to investigate the underlying mechanisms.

Dose- and time-dependent changes in blood and adrenal levels of multiple steroids in rats after administration of ketoconazole with or without ACTH.

To verify simultaneous measurement of blood levels of adrenal steroids as a tool to evaluate drug effects on adrenal steroidogenesis, dose- and time-dependent changes in blood levels of corticosterone and its precursors (pregnenolone, progesterone and deoxycorticosterone), as well as their relationship with the pathological changes in the adrenal gland, were examined in rats dosed with ketoconazole (KET). Also examined were whether effects on adrenal steroidogenesis that were not obvious in the blood steroid levels after sole administration of KET could be revealed by post-administration of ACTH, and the correlation between the blood and adrenal steroid levels. Male rats were dosed with 15, 50, or 150 mg/kg of KET for 1 or 7 days with or without ACTH, and the blood and adrenal concentrations of the steroids were measured. KET increased the blood deoxycorticosterone level even at a dose level and time point at which histopathological changes were not obvious. KET-induced changes in blood levels of other steroids were revealed by ACTH, and the blood and adrenal levels were generally correlated especially after ACTH post-administration. Thus, blood levels of adrenal steroids, including precursors, can be a sensitive and early marker of drug effects on the adrenal steroidogenesis that reflect adrenal levels of steroids. The usefulness of the multiple steroid measurement as a method for mechanism investigation of drug effects on the adrenal gland can be further enhanced by ACTH.

The classic azole antifungal drugs are highly potent endocrine disruptors in vitro inhibiting steroidogenic CYP enzymes at concentrations lower than therapeutic Cmax.

Azole antifungal drugs are used worldwide to treat a variety of fungal infections such as vulvovaginal candidiasis, particularly in pregnant women who are at increased risk. The aim of this study was to mechanistically investigate the endocrine disrupting potential of four commonly used azole antifungal drugs; clotrimazole, miconazole, ketoconazole and fluconazole in vitro using the H295R cell assay and two recombinant, CYP17A1 and CYP19A1 (aromatase), assays. Steroids were quantified using LC-MS/MS. In both recombinant assays, all four azoles inhibited the CYP enzymes investigated, at therapeutically relevant concentrations. However, responses were much more complex in the H295R cell line. Clotrimazole inhibited steroid production in a dose-dependent manner with IC50 values for CYP17A1 and CYP19A1 in the range 0.017-0.184 µM. Miconazole and ketoconazole increased all steroids on the hydroxylase axis (IC50 MIC: 0.042-0.082 µM, KET: 0.041-1.2 µM), leading to accumulation of progestagens and corticosteroids and suppression of androgens and estrogens, indicating inhibition of CYP17A1, in particular lyase activity. However, ketoconazole suppressed all steroids at higher concentrations, resulting in bell-shaped curves for all steroids on the hydroxylase axis. Fluconazole was found to inhibit CYP17A1-lyase activity, causing suppression of androgens (IC50 = 114-209 µM) and estrogens (IC50 = 28 µM). The results indicate that these four azole drugs are highly potent in vitro and, based on plasma Cmax values, may exert endocrine disrupting effects at therapeutically relevant concentrations. This raises concern for endocrine related effects in patients using azole antifungal drugs, particularly when taken during sensitive periods like pregnancy.

Co-exposure to ketoconazole alters effects of bisphenol A in Danio rerio and H295R cells.

Chemicals are present in combination in ambient water, however toxicities of their mixtures are not well understood. This study investigated the effects of ketoconazole (KCZ) on the responses induced by bisphenol A (BPA) in zebrafish and in human adrenocarcinoma (H295R) cells. After exposure to BPA alone or mixed with KCZ for 21 d, egg production, relative tissue weights, sex hormone levels, cytochrome P450 (CYP)3a activity, and transcriptions of genes related to CYP metabolism, vitellogenesis, and steroidogenesis were determined in zebrafish. Male fish were more sensitive to the adverse effects of BPA than females, and the presence of KCZ potentiated the BPA-induced estrogenic responses in the male and anti-estrogenic responses in the female fish. In male zebrafish exposed to BPA, a significant reduction in egg number and relative gonad weight, an increase in 17ß-estradiol (E2) to testosterone (T) ratio, and an upregulation of vtg, erα, and cyp19a genes were observed. Under KCZ, BPA exposure resulted in a significant downregulation of cyp3a65 and pxr genes and an increase in estrogenic responses in males. In female fish, anti-estrogenic effects, such as a decrease in E2 concentration, were observed following the combined exposure. These results indicate that KCZ could increase the toxicity of the chemicals that depend on the given CYP metabolism for their elimination or other crucial functions such as steroidogenesis. Co-exposure to BPA and KCZ in H295R cells also increased E2 and decreased T production. Release and presence of this azole compound warrant caution, because it could modify adverse effects of BPA.

Comparative omics analyses of hepatotoxicity induced by oral azole drugs in mice liver and primary hepatocytes.

Ketoconazole (KTZ) and itraconazole (ITZ) are antifungal agents that have a broad spectrum of activity against fungal pathogens. However, the therapeutic indications of many antifungal drugs, including those of the azole group, are restricted due to possible hepatotoxicity. We performed toxicogenomic analyses using in vivo and in vitro models to investigate the molecular mechanisms underlying the hepatotoxicity of two azole antifungal drugs. C57BL/6 male mice were treated daily with KTZ or ITZ, sacrificed at days 1 or 7, and the serum biochemistry and histopathology results showed that the KTZ-treated mice exhibited hepatotoxicity. Primary hepatocytes from C57BL/6 mice also exposed to KTZ or ITZ, and the cytotoxic effects of KTZ and ITZ were evaluated; KTZ exerted a greater cytotoxic effect than ITZ. The gene expression profiles in the livers of the 7-day-treated group and primary hepatocytes of the 24-h-treated group for both KTZ and ITZ were comparatively analyzed. Differentially expressed genes were selected based on the fold-changes and statistical significance, and the biological functions were analyzed using ingenuity pathways analysis. The results revealed that genes related to cholesterol synthesis were overexpressed in the liver in the KTZ-treated group, whereas expression of those related to acute phase injury was significantly altered in the ITZ-treated group. Causal gene analyses suggested that sterol regulatory element-binding transcription factors are key regulators that activate the transcription of target genes associated with the hepatotoxicity induced by oral KTZ. These findings enhance our understanding of the molecular mechanisms underlying the hepatotoxicity of azole drugs.

Experimental Evidence of Liver Injury by BSEP-Inhibiting Drugs With a Bile Salt Supplementation in Rats.

The bile salt export pump (BSEP, ABCB11) mediates bile acid efflux from hepatocytes into bile. Although the inhibition of BSEP has been implicated as an important mechanism of drug-induced liver injury (DILI), liver injury caused by BSEP-inhibiting drugs is rarely reproduced in experimental animals, probably due to species differences in bile acid composition between humans and rodents. In this study, we tested whether supplementation with chenodeoxycholic acid (CDCA) sodium, a hydrophobic bile salt, could sensitize rats to liver injury caused by a BSEP-inhibiting drug. A potent BSEP inhibitor, ketoconazole (KTZ), which is associated with clinical DILI, was intragastrically administered simultaneously with CDCA at a nontoxic dose once a day for 3 days. Plasma transaminase levels significantly increased in rats receiving CDCA+KTZ, whereas neither treatment with CDCA alone, KTZ alone nor a combination of CDCA and miconazole, a safe analog to KTZ, induced liver injury. In CDCA+KTZ-treated rats, most bile acid species in the liver significantly increased compared with treatment with vehicle or CDCA alone, suggesting that KTZ administration inhibited bile acid excretion. Furthermore, hepatic mRNA expression levels of a bile acid synthesis enzyme, Cyp7a1, and a basolateral bile salt influx transporter, Ntcp, decreased, whereas a canalicular phosphatidylcholine flippase, Mdr2, increased in the CDCA+KTZ group to compensate for hepatic bile acid accumulation. In conclusion, we found that oral CDCA supplementation predisposed rats to KTZ-induced liver injury due to the hepatic accumulation of bile acids. This method may be useful for assessing the potential of BSEP-inhibiting drugs inducing liver injury in vivo.

Mitochondrial Genome Variation Affects Multiple Respiration and Nonrespiration Phenotypes in Saccharomyces cerevisiae.

Mitochondrial genome variation and its effects on phenotypes have been widely analyzed in higher eukaryotes but less so in the model eukaryote Saccharomyces cerevisiae Here, we describe mitochondrial genome variation in 96 diverse S. cerevisiae strains and assess associations between mitochondrial genotype and phenotypes as well as nuclear-mitochondrial epistasis. We associate sensitivity to the ATP synthase inhibitor oligomycin with SNPs in the mitochondrially encoded ATP6 gene. We describe the use of iso-nuclear F1 pairs, the mitochondrial genome equivalent of reciprocal hemizygosity analysis, to identify and analyze mitochondrial genotype-dependent phenotypes. Using iso-nuclear F1 pairs, we analyze the oligomycin phenotype-ATP6 association and find extensive nuclear-mitochondrial epistasis. Similarly, in iso-nuclear F1 pairs, we identify many additional mitochondrial genotype-dependent respiration phenotypes, for which there was no association in the 96 strains, and again find extensive nuclear-mitochondrial epistasis that likely contributes to the lack of association in the 96 strains. Finally, in iso-nuclear F1 pairs, we identify novel mitochondrial genotype-dependent nonrespiration phenotypes: resistance to cycloheximide, ketoconazole, and copper. We discuss potential mechanisms and the implications of mitochondrial genotype and of nuclear-mitochondrial epistasis effects on respiratory and nonrespiratory quantitative traits.

Mechanism-based risk assessment strategy for drug-induced cholestasis using the transcriptional benchmark dose derived by toxicogenomics.

Cholestasis is one of the major causes of drug-induced liver injury (DILI), which can result in withdrawal of approved drugs from the market. Early identification of cholestatic drugs is difficult due to the complex mechanisms involved. In order to develop a strategy for mechanism-based risk assessment of cholestatic drugs, we analyzed gene expression data obtained from the livers of rats that had been orally administered with 12 known cholestatic compounds repeatedly for 28 days at three dose levels. Qualitative analyses were performed using two statistical approaches (hierarchical clustering and principle component analysis), in addition to pathway analysis. The transcriptional benchmark dose (tBMD) and tBMD 95% lower limit (tBMDL) were used for quantitative analyses, which revealed three compound sub-groups that produced different types of differential gene expression; these groups of genes were mainly involved in inflammation, cholesterol biosynthesis, and oxidative stress. Furthermore, the tBMDL values for each test compound were in good agreement with the relevant no observed adverse effect level. These results indicate that our novel strategy for drug safety evaluation using mechanism-based classification and tBMDL would facilitate the application of toxicogenomics for risk assessment of cholestatic DILI.