Me-too validation study for in vitro skin irritation test with a reconstructed human epidermis model, KeraSkin™ for OECD test guideline 439.

We conducted a me-too validation study to confirm the reproducibility, reliability, and predictive capacity of KeraSkin™ skin irritation test (SIT) as a me-too method of OECD TG 439. With 20 reference chemicals, within-laboratory reproducibility (WLR) of KeraSkin™ SIT in the decision of irritant or non-irritant was 100%, 100%, and 95% while between-laboratory reproducibility (BLR) was 100%, which met the criteria of performance standard (PS, WLR≥90%, BLR≥80%). WLR and BLR were further confirmed with intra-class correlation (ICC, coefficients >0.950). WLR and BLR in raw data (viability) were also shown with a scatter plot and Bland-Altman plot. Comparison with existing VRMs with Bland-Altman plot, ICC and kappa statistics confirmed the compatibility of KeraSkin™ SIT with OECD TG 439. The predictive capacity of KeraSkin™ SIT was estimated with 20 reference chemicals (the sensitivity of 98.9%, the specificity of 70%, and the accuracy of 84.4%) and additional 46 chemicals (for 66 chemicals [20 + 46 chemicals, the sensitivity, specificity and accuracy: 95.2%, 82.2% and 86.4%]). The receiver operating characteristic (ROC) analysis suggested a potential improvement of the predictive capacity, especially sensitivity, when changing cut-off (50% → 60-75%). Collectively, the me-too validation study demonstrated that KeraSkin™ SIT can be a new me-too method for OECD TG 439.

A ß-galactosidase-expressing E. coli culture as an alternative test to identify skin sensitizers and non-sensitizers.

Although the Organization for Economic Cooperation and Development (OECD) has adopted several in vitro methods with reasonable predictive capacity, alternative methods for identifying skin sensitizers and non-sensitizers with reliability and simplicity are still required for more efficient and economic prediction. The present study was to design an in vitro system with the use of a ß-galactosidase-expressing E. coli culture for simpler but sufficiently accurate classification of skin sensitizers and non-sensitizers. A LacZ gene-containing E. coli strain that is capable of producing ß-galactosidase enzyme was induced by isopropyl ß-D-1-thiogalactopyranoside with concomitant treatment with test chemicals. After 6-hr incubation, cells were lysed and ß-galactosidase enzyme activity was monitored colorimetrically by using O-nitrophenyl-D-galactopyranoside as a substrate. Following optimization of several experimental conditions, 22 skin sensitizers and 11 non-sensitizers were examined to assess predictive capacity of this method. The results indicated that predictivity was as follows: 90.9% sensitivity, 81.8% specificity, and 87.9% accuracy, when 17.3% of control activity was used as the cut-off value to separate sensitizers from non-sensitizers. Data suggested that the current bacterial system expressing ß-galactosidase may serve as a useful alternative test for classifying skin sensitizers and non-sensitizers, without the utilization of animals or mammalian cell cultures.

Role of Intestinal Microbiota in Baicalin-Induced Drug Interaction and Its Pharmacokinetics.

Since many glycoside compounds in natural products are hydrolyzed by intestinal microbiota when administered orally, it is of interest to know whether their pharmacological effects are derived from the glycoside itself or from the aglycone form in vivo. An interesting example is baicalin versus baicalein, the aglycone of baicalin, which is contained in some herbs from Labiatae including Scutellaria baicalensis Georgi and Scutellaria lateriflora Linne. The herbs have been extensively used for treatment of inflammatory diseases in Asia. Although there have been numerous reports regarding the pharmacological effects of baicalin and baicalein in vivo and in vitro, some reports indicated that the glycoside form would hardly be absorbed in the intestine and that it should be hydrolyzed to baicalein in advance for absorption. Therefore, the role of metabolism by intestinal microbiota should also be considered in the metabolism of baicalin. In addition, baicalin contains a glucuronide moiety in its structure, by which baicalin and baicalein show complex pharmacokinetic behaviors, due to the interconversion between them by phase II enzymes in the body. Recently, concerns about drug interaction with baicalin and/or baicalein have been raised, because of the co-administration of Scutellaria species with certain drugs. Herein, we reviewed the role of intestinal microbiota in pharmacokinetic characteristics of baicalin and baicalein, with regards to their pharmacological and toxicological effects.

Nephrotoxic potential and toxicokinetics of melamine combined with cyanuric acid in rats.

To investigate the nephrotoxic potential of melamine (MEL) and cyanuric acid (CA) in male Sprague-Dawley rats, 7-d repeated-dose studies were performed. The experimental groups of MEL100 and CA100 were orally administered with MEL and CA at 100 mg/kg/d for 7 d, respectively. In groups dosed with MEL-CA mixtures, melamine and cyanuric acid (1:1) were simultaneously administered at 4, 20, or 100 mg/kg/d for 7 d (i.e., MEL-CA4, MEL-CA20, or MEL-CA100, respectively). Body weights were not markedly affected in MEL100, CA100, and MEL-CA4 groups, but significantly reduced in MEL-CA 20 and 100 rats. Most parameters determined in sera and tissues were not markedly altered in MEL100, CA100, and MEL-CA4-treated rodents. However, BUN, creatinine, total protein, and kidney weights were significantly increased in MEL-CA20- and MEL-CA100-treated animals. Renal histopathologic findings also revealed signs of toxicity, including tubular dilatation, crystal deposition, granulomatous tubulo-interstitial inflammation, and tubular necrosis with regeneration. Data suggested that the combination of MEL and CA might be responsible for observed nephrotoxicity that was not seen following individual exposure to either MEL or CA alone. Subsequently, the concentrations of MEL and CA were determined in serum, urine, and kidney tissues by using liquid chromatography-mass spectrometry. Toxicokinetic studies indicated that MEL or CA alone might be eliminated almost completely within 24 h after dosing showing no accumulation in kidney. However, the combined MEL-CA dose produced marked accumulation of chemicals in blood and kidneys. These results suggested that combined MEL and CA might produce renal toxicity due to significant chemical accumulation in kidney accompanied by low excretion.

Role of metabolism by the human intestinal microflora in arbutin-induced cytotoxicity in HepG2 cell cultures.

A possible role for metabolism by the human intestinal microflora in arbutin-induced cytotoxicity was investigated using human hepatoma HepG2 cells. When the cytotoxic effects of arbutin and hydroquinone (HQ), a deglycosylated metabolite of arbutin, were compared, HQ was more toxic than arbutin. Incubation of arbutin with a human fecal preparation could produce HQ. Following incubation of arbutin with a human fecal preparation for metabolic activation, the reaction mixture was filter-sterilized to test its toxic effects on HepG2 cells. The mixture induced cytotoxicity in HepG2 cells in a concentration-dependent manner. In addition, the mixture considerably inhibited expression of Bcl-2 together with an increase in Bax expression. Likewise, activation stimulated cleavage of caspase-3 and production of reactive oxygen species in HepG2 cell cultures. Furthermore, induction of apoptosis by the intestinal microflora reaction mixture was confirmed by the terminal deoxynucleotidyltransferase-mediated dUTP-biotin nick-end labeling assay. Taken together, these findings suggest that the human intestinal microflora is capable of metabolizing arbutin to HQ, which can induce apoptosis in mammalian cells.

ChemSkin Reference Chemical Database for the Development of an In Vitro Skin Irritation Test.

Since the animal test ban on cosmetics in the EU in 2013, alternative in vitro safety tests have been actively researched to replace in vivo animal tests. For the development and evaluation of a new test method, reference chemicals with quality in vivo data are essential to assess the predictive capacity and applicability domain. Here, we compiled a reference chemical database (ChemSkin DB) for the development and evaluation of new in vitro skin irritation tests. The first candidates were selected from 317 chemicals (source data n = 1567) searched from the literature from the last 20 years, including previous validation study reports, ECETOC, and published papers. Chemicals showing inconsistent classification or those that were commercially unavailable, difficult or dangerous to handle, prohibitively expensive, or without quality in vivo or in vitro data were removed, leaving a total of 100 chemicals. Supporting references, in vivo Draize scores, UN GHS/EU CLP classifications and commercial sources were compiled. Test results produced by the approved methods of OECD Test No. 439 were included and compared using the classification table, scatter plot, and Pearson correlation analysis to identify the false predictions and differences between in vitro skin irritation tests. These results may provide an insight into the future development of new in vitro skin irritation tests.

Synthesis, topoisomerase I and II inhibitory activity, cytotoxicity, and structure-activity relationship study of hydroxylated 2,4-diphenyl-6-aryl pyridines.

A new series of 2,4-diphenyl-6-aryl pyridines containing hydroxyl group(s) at the ortho, meta, or para position of the phenyl ring were synthesized, and evaluated for topoisomerase I and II inhibitory activity and cytotoxicity against several human cancer cell lines for the development of novel anticancer agents. Structure-activity relationship study revealed that the substitution of hydroxyl group(s) increased topoisomerase I and II inhibitory activity in the order of meta > para > ortho position. Substitution of hydroxyl group on the para position showed better cytotoxicity.

Role of metabolism in 1-bromopropane-induced hepatotoxicity in mice.

A possible role of metabolism in 1-bromopropane (1-BP)-induced hepatotoxicity was investigated in male ICR mice. The depletion of glutathione (GSH) by formation of GSH conjugates was associated with increased hepatotoxicity in 1-BP-treated mice. The formation of S-propyl and 2-hydroxypropyl GSH conjugates were identified in the liver following 1-BP treatment. In addition, the formation of reactive metabolites of 1-BP by certain cytochrome P-450 (CYP) may be involved in 1-BP-induced hepatotoxicity. The decreased content of hepatic GSH produced by 1-BP was associated not only with increased activities of alanine aminotransferase (ALT) and aspartate aminotransferase (AST) but also with elevated levels of hepatic thiobarbituric acid-reactive substance (TBARS) in mice where metabolic enzymes were induced by pretreatment with phenobarbital. In addition, the hepatotoxicity induced by 1-BP was prevented by pretreatment with SKF-525A. Taken together, the formation of reactive metabolites by CYP and depletion of GSH may play important roles in hepatotoxicity induced by 1-BP.

Inhibitory effects of deoxypodophyllotoxin from Anthriscus sylvestris on human CYP2C9 and CYP3A4.

Deoxypodophyllotoxin (DPT) is a bioactive compound of Anthriscus sylvestris (Apiaceae). In the present study, the inhibition of cytochrome P450 (CYP) by DPT was evaluated in human liver microsomes (HLM) and the baculovirus-insect cell-expressed human CYPs using a cocktail probe assay. When a mixture of specific CYP substrates was incubated with DPT in HLM, CYP2C9-catalyzed diclofenac 4-hydroxylation and CYP3A4-catalyzed midazolam 1-hydroxylation were strongly inhibited by DPT, with IC (50) values of 6.3 and 9.2 microM, respectively. The Lineweaver-Burke plots for the inhibition of CYP2C9 and CYP3A4 in HLM and baculovirus-insect cell-expressed human CYPs were consistent with a competitive type of inhibition. From these results, DPT was characterized to be a competitive inhibitor of CYP2C9 and CYP3A4, with K(i) values of 3.5 and 10.8 microM in HLM and 24.9 and 3.5 microM in baculovirus-insect cell-expressed human CYPs, respectively.

Effects of Intestinal Microbiota on Pharmacokinetics of Crocin and Crocetin in Male Sprague-Dawley Rats.

In addition to the hepatic metabolism, the role of intestinal microbiota in drug metabolism has been considered important in the biotransformation of xenobiotics. Crocin and its aglycone, crocetin, isolated from many plants, including the dried stigma of Crocus sativus and the fruit of Gardenia jasminoides, have been used in treatment of inflammation, cancer, and metabolic disorders. In this study, the effect of intestinal microbiota on the pharmacokinetics of crocin was studied following single oral treatment with 600 mg/kg crocin to male rats pre-treated with a mixture of antibiotics, such as cefadroxil, oxytetracycline, and erythromycin, for three consecutive days. Following crocin treatment, blood, urine, and feces were collected at various time points for evaluating pharmacokinetic characteristics of crocin and crocetin by using LC-MS. Results showed that intestinal absorption of crocin was relatively marginal when compared with that of crocetin, and that crocin metabolism to crocetin by intestinal microbiota would be a critical step for absorption. The present results clearly suggested that the in vivo pharmacological effects of crocin might be considered as the effects by its aglycone, crocetin, mainly, and that the metabolism of glycosidic natural products by intestinal microbiota should be considered to understand their pharmacodynamic actions.

Role of Intestinal Microbiota in Metabolism of Voglibose In Vitro and In Vivo.

BACKGROUND: Voglibose, an α-glucosidase inhibitor, inhibits breakdown of complex carbohydrates into simple sugar units in intestine. Studies showed that voglibose metabolism in the liver might be negligible due to its poor intestinal absorption. Numerous microorganisms live in intestine and have several roles in metabolism and detoxification of various xenobiotics. Due to the limited information, the possible metabolism of voglibose by intestinal microbiota was investigated in vitro and in vivo. METHODS: For the in vitro study, different concentrations of voglibose were incubated with intestinal contents, prepared from both vehicle- and antibiotics-treated mice, to determine the decreased amount of voglibose over time by using liquid chromatography-mass spectrometry. Similarly, in vivo pharmacodynamic effect of voglibose was determined following the administration of voglibose and starch in vehicle- and antibiotic-pretreated non-diabetic and diabetic mice, by measuring the modulatory effects of voglibose on blood glucose levels. RESULTS: The in vitro results indicated that the remaining voglibose could be significantly decreased when incubated with the intestinal contents from normal mice compared to those from antibiotic-treated mice, which had less enzyme activities. The in vivo results showed that the antibiotic pretreatment resulted in reduced metabolism of voglibose. This significantly lowered blood glucose levels in antibiotic-pretreated mice compared to the control animals. CONCLUSION: The present results indicate that voglibose would be metabolized by the intestinal microbiota, and that this metabolism might be pharmacodynamically critical in lowering blood glucose levels in mice.

Nephrotoxic potential and toxicokinetics of tetrabromobisphenol A in rat for risk assessment.

Tetrabromobisphenol A (TBBPA), one of the most widely used global brominated flame retardants, is used to improve fire safety of laminates in electrical and electronic equipment. To investigate the nephrotoxic potential of TBBPA and its toxicokinetic profile in rats, single-dose and daily 14-d repeated-dose toxicity studies at 200, 500, or 1000 mg/kg were performed. Several biochemical parameters were analyzed to evaluate nephrotoxicity of TBBPA. High-dose 1000 mg/kg TBBPA significantly elevated renal thiobarbituric acid-reactive substance (TBARS) levels, and superoxide dismutase (SOD) activity was increased at all 3 doses administered. This was associated with no change in the activity of catalase (CAT). Our results suggest that acute 1-d high-dose administration of TBBPA produced transient renal changes at 5 h. Subsequently, TBBPA in serum, urine, and kidney was determined by liquid chromatography-mass spectroscopy (LC/MS). Toxicokinetic studies indicated that TBBPA shows relatively a short half-life (7-9 h) and was eliminated almost completely in feces by 2 d. Based on the results from the 14-d repeated-dose study, TBBPA did not accumulate in the rat, and was eliminated in feces. The present results suggested that TBBPA may not be toxic to kidney, as the chemical is not bioavailable and is not present in renal tissue.

Identification of pre- and pro-haptens with a ß-galactosidase-expressing E. coli culture system for skin sensitization.

Although several in vitro approaches were successful in separating chemicals as skin sensitizers and non-sensitizers, none of the available methods completely mimics the absolute in vivo scenario of skin sensitization. One of the major challenges with currently available systems would be the limited or no metabolic capacity to activate pre- or pro-haptens to reactive metabolites in the system. In the present study, E. coli cells with ß-galactosidase-expressing LacZ gene were combined with either induced rat liver S-9 fractions or microsomal fractions to detect pre- or pro-haptens to cause skin sensitization. Following optimization of some experimental conditions, we examined 20 sensitizers classified as pre- or pro-haptens and 11 non-sensitizers in these E. coli cultures by incubating bacterial cells and test chemicals with and without S-9 or microsomal proteins. After a 6-h incubation in the presence of IPTG, cells were lyzed to determine the suppression of ß-galactosidase enzyme. A cut-off of 17.3% was applied to determine the percent suppression of ß-galactosidase activity by test chemicals to classify skin sensitizers and non-sensitizers. Among chemicals tested, 19 pre- or pro-haptens were categorized as true positives and 8 non-sensitizers were categorized as true negatives. Thereby, the overall sensitivity, specificity and accuracy achieved with microsome-incorporated and S-9 fraction-incorporated group were 95.0%, 72.7% and 87.1% and 80.0%, 81.8% and 80.6%, respectively. The results suggested that the present bacterial system incorporated with the microsomal activation system could be considered as a useful alternative method to classify not only direct-acting sensitizers but also pre- or pro-haptens requiring metabolic activation in vitro.

Role of Intestinal Microbiota in Metabolism of Gastrodin In Vitro and In Vivo.

Alteration in the number and composition of intestinal microbiota affects the metabolism of several xenobiotics. Gastrodin, isolated from Gastrodia elata, is prone to be hydrolyzed by intestinal microbiota. In the present study, the role of intestinal microbiota in gastrodin metabolism was investigated in vitro and in vivo. Gastrodin was incubated in an anaerobic condition with intestinal contents prepared from vehicle- and antibiotics-treated rats and the disappearance of gastrodin and formation of 4-hydroxybenzyl alcohol (4-HBA) was measured by liquid chromatography coupled to mass spectroscopy (LC-MS/MS). The results showed that almost all gastrodin incubated with control intestinal contents was metabolized to its aglycone in time- and concentration-dependent manners. In contrast, much less formation of 4-HBA was detected in intestinal contents from antibiotics-treated rats. Subsequently, in vivo pharmacokinetic study revealed that the antibiotic pretreatment of rats significantly affected the metabolism of gastrodin to 4-HBA. When administered orally, gastrodin was rapidly absorbed rapidly into plasma, metabolized to 4-HBA, and disappeared from the body within six hours. Interestingly, the pharmacokinetic parameters of 4-HBA were changed remarkably in antibiotics-treated rats, compared to control rats. The results clearly indicated that the antibiotics treatment of rats suppressed the ability of intestinal microbiota to metabolize gastrodin to 4-HBA and that, thereby, the pharmacodynamic action was significantly modulated.

Me-too validation study for in vitro eye irritation test with 3D-reconstructed human cornea epithelium, MCTT HCETM.

The need for in vitro eye irritation test replacing in vivo is steadily increasing. The MCTT HCE™ eye irritation test (EIT) using 3D reconstructed human cornea-like epithelium, was developed to identify ocular irritants from non-irritants those that are not requiring classification and labelling for eye irritation. Here, we report the results of me-too validation study, which was conducted to evaluate the reliability and relevance of the MCTT HCETM EIT, according to performance standards (PS) of OECD TG 492. The optimal cutoff to determine irritation in the prediction model was preliminarily established at 45% with the receiver operation characteristics (ROC) curve for 141 reference substances. To demonstrate the reproducibility of within- and between-laboratory (WLR and BLR), a set of 30 PS reference chemicals were tested in three laboratories three times. The WLR and BLR concordance with the binary decision of whether non-irritant or irritant was estimated to be 90-100% and 90%, respectively, and both met the PS requirements. The predictive capacity of the respective laboratories for the 30 reference chemicals were evaluated based on three different estimation methods, and the results were comparable, with sensitivity ranging from 89.6 to 93.3%, the specificity ranging from 62.2 to 66.7%, and the accuracy ranging from 75.9 to 80.0%. Additional test with the new set of 30 PS substances in the revised OECD GD 216 yielded a performance of sensitivity ranging from 92.6-93.3%, the specificity 62.2-66.7% and the accuracy 77.4-80.0%. 95.0% sensitivity, 67.2% specificity, and 83.0% accuracy were obtained for 141 reference substances in total. Furthermore, separate cutoffs for liquids and solids, 35% and 60%, respectively, produced better predictivity, which was established as a final prediction model. Collectively, our study demonstrated that MCTT HCETM EIT meets the reproducibility and predictivity criteria stated in OECD TG 492 PS.

Identification of glutathione conjugates of 1-bromopentane and its hepatotoxicity in female BALB/c mice.

Halogenated organic compounds, such as 1-bromopentane (1-BPT), are used as cleaning agents, synthesis agents, or extraction solvents in the workplace. In the present study, glutathione (GSH) conjugation and hepatotoxicity induced by 1-BPT were investigated in female BALB/c mice. S-Bromopentyl GSH, S-bromopentyl cysteine, and mono-hydroxypentyl mercapturic acid were identified in liver by liquid chromatography-electrospray ionization tandem mass spectrometry. Oral treatment of mice with 1-BPT at 1500 mg/kg produced maximum GSH conjugates at 6 h after treatment. For hepatotoxicity tests, the animals were treated orally with 1-BPT at 375, 750, or 1500 mg/kg in corn oil once for a dose response study or at 1500 mg/kg for 6, 12, 24, or 48 h for a time course study. 1-BPT dose-dependently increased serum activity of ALT and AST and decreased hepatic GSH levels, peaking at 6 and 12 h after treatment. 1-BPT (750 and 1500 mg/kg) also significantly increased the hepatic content of malondialdehyde. Thus, 1-BPT could cause hepatotoxicity and depletion of GSH content by forming GSH conjugates, presenting a toxicity mechanism and potential biomarkers for low molecular weight haloalkanes.

In vitro characterization of the enzymes involved in the metabolism of 1-furan-2-yl-3-pyridin-2-yl-propenone, an anti-inflammatory propenone compound.

Carbonyl reduction is a significant step in the phase I biotransformation of a great variety of aromatic, alicyclic and aliphatic carbonyl compounds. 1-Furan-2-yl-3-pyridin-2-yl-propenone (FPP-3) has been shown to have anti-inflammatory activity as it inhibits the production of nitric oxide and tumor necrosis factor-beta. In the present study, the metabolic fate and possible involvement of 11beta-hydroxysteroid dehydrogenase (11beta-HSD) and carbonyl reductase (CBR) in the metabolism of FPP-3 were investigated in rat liver subcellular fractions. When FPP-3 was incubated with rat liver subcellular fractions in the presence of beta-NADPH, two major peaks were detected by reduction on the propenone: M1 (1-furan-2-yl-3-pyridin-2-yl-propan-1-one) and M2 (1-furan-2-yl-3-pyridin-2-yl-propan-1-ol). Inhibitors of CBR, such as quercitrin, ethacrynic acid and menadione, significantly increased the formation of M1, but effectively inhibited the formation of M2 in subcellular fractions. Meanwhile, 18beta-glycyrrhetinic acid, a selective inhibitor of 11beta-HSD, marginally inhibited the reduction of FPP-3 in microsomes. A good correlation was observed between the formation of M2 and CBR activity with either 4-pyridine carboxaldehyde (r=0.72) or D,L-glyceraldehyde (r=0.63) as substrates in the cytosol. These results indicated that FPP-3 might be metabolized by cytosolic CBR and uncharacterized microsomal reductase(s) in rat liver.

A simple in chemico method for testing skin sensitizing potential of chemicals using small endogenous molecules.

Among many of the validated methods for testing skin sensitization, direct peptide reactivity assay (DPRA) employs no cells or animals. Although no immune cells are involved in this assay, it reliably predicts the skin sensitization potential of a chemical in chemico. Herein, a new method was developed using endogenous small-molecular-weight compounds, cysteamine and glutathione, rather than synthetic peptides, to differentiate skin sensitizers from non-sensitizers with an accuracy as high as DPRA. The percent depletion of cysteamine and glutathione by test chemicals was measured by an HPLC equipped with a PDA detector. To detect small-size molecules, such as cysteamine and glutathione, a derivatization by 4-(4-dimethylaminophenylazo) benzenesulfonyl chloride (DABS-Cl) was employed prior to the HPLC analysis. Following test method optimization, a cut-off criterion of 7.14% depletion was applied to differentiate skin sensitizers from non-sensitizers in combination of the ratio of 1:25 for cysteamine:test chemical with 1:50 for glutathione:test chemical for the best predictivity among various single or combination conditions. Although overlapping HPLC peaks could not be fully resolved for some test chemicals, high levels of sensitivity (100.0%), specificity (81.8%), and accuracy (93.3%) were obtained for 30 chemicals tested, which were comparable or better than those achieved with DPRA.

Exploring the Metabolism of Loxoprofen in Liver Microsomes: The Role of Cytochrome P450 and UDP-Glucuronosyltransferase in Its Biotransformation.

Loxoprofen, a propionic acid derivative, non-steroidal anti-inflammatory drug (NSAID) is a prodrug that is reduced to its active metabolite, trans-alcohol form (Trans-OH) by carbonyl reductase enzyme in the liver. Previous studies demonstrated the hydroxylation and glucuronidation of loxoprofen. However, the specific enzymes catalyzing its metabolism have yet to be identified. In the present study, we investigated metabolic enzymes, such as cytochrome P450 (CYP) and UDP-glucuronosyltransferase (UGT), which are involved in the metabolism of loxoprofen. Eight microsomal metabolites of loxoprofen were identified, including two alcohol metabolites (M1 and M2), two mono-hydroxylated metabolites (M3 and M4), and four glucuronide conjugates (M5, M6, M7, and M8). Based on the results for the formation of metabolites when incubated in dexamethasone-induced microsomes, incubation with ketoconazole, and human recombinant cDNA-expressed cytochrome P450s, we identified CYP3A4 and CYP3A5 as the major CYP isoforms involved in the hydroxylation of loxoprofen (M3 and M4). Moreover, we identified that UGT2B7 is the major UGT isoform catalyzing the glucuronidation of loxoprofen and its alcoholic metabolites. Further experimental studies should be carried out to determine the potency and toxicity of these identified metabolites of loxoprofen, in order to fully understand of mechanism of loxoprofen toxicity.

Intra- and inter-laboratory reproducibility and predictivity of the HaCaSens assay: A skin sensitization test using human keratinocytes, HaCaT.

Due to considerable constraints in using animals for risk assessment, much effort has been directed at developing non-animal test methods. Developing assays for skin sensitization, the leading cause of contact dermatitis, is particularly important, but there are currently no in vitro skin sensitization tests that completely replace animal tests. HaCaSens, a simple skin sensitization test using non-transformed HaCaT cells, predicts keratinocyte activation by skin sensitizers with 75% sensitivity, 83% specificity and 77% accuracy in a previous study using 22 coded substances. Although the data show promising results, the number of tested substances is insufficient to prove predictive capacity. Moreover, reproducibility among different laboratories has not been studied. Here, three laboratories participated in a validation in order to assess HaCaSens feasibility for official validation. To examine transferability, intra- and inter-lab reproducibility and predictive capacity, HaCaSens was assessed on a set of 30 test substances coordinated by the Validation Management Team (VMT). The results showed satisfactory transferability as well as intra- and inter-laboratory reproducibility. Further assessment of its predictive capacity on 20 test substances demonstrated a sensitivity of 81.8% (18/22), specificity of 87.5% (7/8), and accuracy of 83.3% (25/30) in identifying skin sensitizers, which is comparable with presently validated assays, KeratinoSens™ and LuSens. This validation study shows that the HaCaSens assay is easily transferable, reproducible and highly predictable for identifying skin sensitizers.