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Graphical abstract, Highlights • Permeability values of 90 industry chemicals were measured by a Caco-2 system. • A multivariate prediction equation for permeability of chemicals was proposed. • Chemical permeability coefficients were inversely associated with hepatic NOELs., Apparent permeability coefficients (Papp) across a human intestinal epithelial Caco-2 cell monolayer were measured for a range of industrial/drug chemicals. A predictive equation for determining in vitro Papp values of fifty-six substances was set up using multivariate regression analysis based on in silico-estimated physicochemical properties (molecular weights and water distribution coefficients for apical and basal pH environments) (r = 0.77, p

Prostacyclin alternatively called prostaglandin (PG) I2 is an unstable metabolite synthesized by the arachidonate cyclooxygenase pathway. Earlier studies have suggested that prostacyclin analogues can act as a potent effector of adipose differentiation. However, biosynthesis of PGI2 has not been determined comprehensively at different life stages of adipocytes. PGI2 is rapidly hydrolyzed to the stable product, 6-keto-PGF1α, in biological fluids. Therefore, the generation of PGI2 can be quantified as the amount of 6-keto-PGF1α. In this study, we attempted to develop a solid-phase enzyme-linked immunosorbent assay (ELISA) using a mouse antiserum specific for 6-keto-PGF1α. According to the typical calibration curve of our ELISA, 6-keto-PGF1α can be quantified from 0.8 pg to 7.7 ng in an assay. The evaluation of our ELISA revealed the higher specificity of our antiserum without the cross-reaction with other related prostanoids while it exhibited only the cross-reaction of 1.5 % with PGF2α. The resulting ELISA was applied to the quantification of 6-keto-PGF1α generated endogenously by cultured 3T3-L1 cells at different stages. The cultured cells showed the highest capability to generate 6-keto-PGF1α during the maturation phase of 4–6 days, which was consistent with the coordinated changes in the gene expression of PGI synthase and the IP receptor for PGI2. Following these events, the accumulation of fats was continuously promoted up to 14 days. Thus, our immunological assay specific for 6-keto-PGF1α is useful for monitoring the endogenous levels of the unstable parent PGI2 at different life stages of adipogenesis and for further studies on the potential association with the up-regulation of adipogenesis in cultured adipocytes.

We have previously shown that cultured adipocytes have the ability to biosynthesize prostaglandin (PG) I2 called alternatively as prostacyclin during the maturation phase by the positive regulation of gene expression of PGI synthase and the prostanoid IP receptor. To clarify how prostacyclin regulates adipogenesis, we investigated the effects of prostacyclin and the specific agonists or antagonists for the IP receptor on the storage of fats during the maturation phase of cultured adipocytes. Exogenous PGI2 and the related selective agonists for the IP receptor including MRE-269 and treprostinil rescued the storage of fats attenuated by aspirin, a cyclooxygenase inhibitor. On the other hand, selective antagonists for IP such as CAY10441 and CAY10449 were effective to suppress the accumulation of fats as GW9662, a specific antagonist for peroxisome proliferator-activated receptor (PPAR)γ. Thus, pro-adipogenic action of prostacyclin can be explained by the action mediated through the IP receptor expressed at the maturation stage of adipocytes. Cultured adipocytes incubated with each of PGI2 and MRE-269 together with troglitazone, an activator for PPARγ, exhibited additively higher stimulation of fats storage than with either compound alone. The combined effect of MRE-269 and troglitazone was almost abolished by co-incubation with GW9662, but not with CAY10441. Increasing concentrations of troglitazone were found to reverse the inhibitory effect of CAY10441 in a dose-dependent manner while those of MRE-269 failed to rescue adipogenesis suppressed by GW9662, indicating the critical role of the PPARγ activation as a downstream factor for the stimulated adipogenesis through the IP receptor. Treatment of cultured adipocytes with cell permeable stable cAMP analogues or forskolin as a cAMP elevating agent partly restored the inhibitory effect of aspirin. However, excess levels of cAMP stimulated by forskolin attenuated adipogenesis. Supplementation with H-89, a cell permeable inhibitor for protein kinase A (PKA), had no effect on the promoting action of PGI2 or MRE-269 along with aspirin on the storage of fats, suggesting that the promotion of adipogenesis mediated by the IP receptor does not require the PKA activity.

Human biomonitoring of plasma and urinary levels of nicotine, cotinine, and 3′-hydroxycotinine was conducted after daily cigarette smoking in a population of 92 male Japanese smokers with a mean age of 37 years who had smoked an average of 23 cigarettes per day for 16 years. Members of the population were genotyped for the nicotine-metabolizing enzyme cytochrome P450 2A6 (CYP2A6). The mean levels of nicotine, the levels of its metabolites cotinine and 3′-hydroxycotinine, and the sum of these three levels in subjects one hour after smoking the first cigarette on the sampling day were 20.1, 158, 27.7, and 198 ng/mL in plasma and 846, 1,020, 1,010, and 2,870 ng/mL in urine under daily smoking conditions. Plasma levels of 3'-hydroxycotinine and urinary levels of nicotine and 3′-hydroxycotinine were dependent on the CYP2A6 phenotype group, which was estimated from the CYP2A6 genotypes of the subjects, including those with whole gene deletion. Plasma cotinine levels were significantly correlated with the number of cigarettes smoked on the day before sampling (r = 0.71), the average number of cigarettes smoked daily (r = 0.58), and the Brinkman index (daily cigarettes × years, r = 0.48) under the present conditions. The sum of nicotine, cotinine, and 3′-hydroxycotinine concentrations in plasma showed a similar relationship to that of the plasma cotinine levels. Urinary concentrations of cotinine and the sum of nicotine metabolite concentrations also showed significant correlations with the plasma levels and the previous day’s and average cigarette consumption. The numbers of cigarettes smoked per day by two subjects with self-reported light smoking habits were predicted by measuring the urinary cotinine concentrations and using linear regression equations derived from above-mentioned data. These results indicate that biomonitoring of the urinary cotinine concentration is a good, easy-to-use marker for plasma levels of cotinine and the sum of nicotine metabolites in smokers independent of genetic polymorphism of CYP2A6.

The present study defined a simplified physiologically based pharmacokinetic (PBPK) model for nicotine and its primary metabolite cotinine in humans, based on metabolic parameters determined in vitro using relevant liver microsomes, coefficients derived in silico, physiological parameters derived from the literature, and an established rat PBPK model. The model consists of an absorption compartment, a metabolizing compartment, and a central compartment for nicotine and three equivalent compartments for cotinine. Evaluation of a rat model was performed by making comparisons with predicted concentrations in blood and in vivo experimental pharmacokinetic values obtained from rats after oral treatment with nicotine (1.0 mg/kg, a no-observed-adverse-effect level) for 14 days. Elimination rates of nicotine in vitro were established from data from rat liver microsomes and from human pooled liver microsomes. Human biomonitoring data (17 ng nicotine and 150 ng cotinine per mL plasma 1 h after smoking) from pooled five male Japanese smokers (daily intake of 43 mg nicotine by smoking) revealed that these blood concentrations could be calculated using a human PBPK model. These results indicate that a simplified PBPK model for nicotine/cotinine is useful for a forward dosimetry approach in humans and for estimating blood concentrations of other related compounds resulting from exposure to low chemical doses.

A sensitive and reliable high-pressure liquid chromatography (HPLC) assay for miloxacin and its two principal metabolites, 5,8-dihydro-8-oxo-2H-1,3-dioxolo[4,5-g]quinoline-7-carboxylic acid (M-1) and 1,4-dihydro-1,6-dimethoxy-7-hydroxy-4-oxoquinoline-3-carboxylic acid (M-2), in human serum and urine was developed. A strong anion-exchange Zipax SAX column using a mobile phase of 0.01 M citric acid solution containing 0.03 M sodium nitrate with pH 5.0 was used to achieve separation of the three compounds. The retention times of miloxacin, M-1, and M-2 were 3.8, 9.3, and 5.9 min, respectively. Serum and urine concentrations of these compounds as low as 10 ng/ml were measured. When results from the HPLC assay were compared with those from the microbiological assay of serum and urine samples from human subjects receiving miloxacin orally, the correlation coefficients were 0.94 for the serum and 0.99 for the urine. The HPLC assay method presents an alternative to the microbiological assay and permits future pharmacokinetic investigations of miloxacin.