Synthesis, design, and antiproliferative evaluation of 6-(N-Substituted-methyl)pyrazolo[3,4-d]pyrimidines as the potent anti-leukemia agents.

Pyrazolopyrimidine derivatives, including pyrazolopyrimidines, 6-aminopyrazolopyrimidines, 6-[(formyloxy)methyl]pyrazolopyrimidines, 6-(hydroxymethyl)pyrazolopyrimidine, and 6-(aminomethyl)pyrazolopyrimidines have been successfully prepared and tested against NCI-H226, NPC-TW01, and Jurkat cancer cell lines. Among the tested pyrazolopyrimidine compounds, we found 6-aminopyrazolopyrimidines and 6-(aminomethyl)pyrazolopyrimidines with essential o-ClPh or p-ClPh substituted moieties on N-1 pyrazole ring exhibited the best IC50 inhibition activity for Jurkat cells. Furthermore, optimization of the SAR study on the C-6 position of pyrazolopyrimidine ring demonstrated that 6-(N-substituted-methyl)pyrazolopyrimidines 17b, 17d, and 19d possessed the significant IC50 inhibitory activity for the different leukemia cell lines, especially for Jurkat, K-562, and HL-60. On the other hand, further SAR inhibition and docking model studies revealed that compound 19d, which has a 3-(1H-imidazol-1-yl)propan-1-amino side-chain on the C-6 position, was able to form four hydrogen bonds with residues Ala226, Leu152, and Glu194 and specifically extended into the P1 pocket subsite with Aurora A, resulting in improved inhibitory activity almost similar to SNS-314. To explore the anti-cancer mechanism, compound 19d was measured by Western blot analysis in Jurkat T-cells, however, it showed non-responsibility to Aurora B. For the further structural modifications on the lateral chain of compound 19d, compounds 24 with longer lateral chain were designed and synthesized for testing leukemia cell lines. However, compounds 24 was significantly decrease inhibition potency against leukemia cell lines. Based on the in-vitro results, compounds 17b and 19d could be considered to be the best potential lead drug in our study for the development of new and effective therapies for leukemia treatment. On the other hand, the DHFR inhibition results indicated compound 19d possessed good inhibitory activity and better than the reported naphthalene derivative. Through further comparisons of the model superposition of three-dimensional (3D) conformations in DHFR, compound 19d presented a similar structural alignment to Methotrexate and the reported naphthalene derivative and led to similar drug-like functional relationships. As a results, compound 19d would be a potential DHFR inhibitor for anti-leukemia drug candidate.

Synthesis, physicochemical characterization, and investigation of anti-inflammatory activity of water-soluble PEGylated 1,2,4-Triazoles.

A series of water-soluble PEGylated 1,2,4-triazoles 5-8 were successfully synthesized from methyl 5-(chloromethyl)-1-aryl-1H-1,2,4-triazole-3-carboxylates 1. All of the water-soluble PEGylated 1,2,4-triazoles were characterized by FT-IR and 1H NMR spectroscopy. The solubility, in vitro plasma stability, and anti-inflammatory activity were also determined and compared to original methyl 5-(halomethyl)-1-aryl-1H-1,2,4-triazole-3-carboxylates. For SAR study, all PEGylated 1,2,4-triazoles 5-8 performed potential anti-inflammatory activity on LPS-induced RAW 264.7 cells (IC50 = 3.42-7.81 µM). Moreover, the western blot result showed PEGylated 1,2,4-triazole 7d performed 5.43 and 2.37 folds inhibitory activity over iNOS and COX-2 expressions. On the other hand, the cell viability study revealed PEGylated 1,2,4-triazoles 7 and 8 with PEG molecular weight more than 600 presented better cell safety (cell viability > 95 %). Through the solubility and in vitro plasma stability studies, PEGylated 1,2,4-triazoles 7a-d exhibited higher hydrophilicity and prolonged 2.01 folds of half-life in compound 7d. Furthermore, the in vivo anti-inflammatory and gastric safety results indicated PEGylated 1,2,4-triazole 7d more effectively decreased the inflammatory response in edema and COX-2 expression and exhibited higher gastric safety than Indomethacin. Following the in vitro and in vivo study results, PEGylated 1,2,4-triazole 7d possessed favorable solubility, plasma stability features, safety, and significant anti-inflammatory activity to become the potential water-soluble anti-inflammatory candidate.

2-Chloro-3,7,8-tribromodibenzofuran as a new environmental pollutant inducing atypical ultrasonic vocalization in infant mice.

Epidemiological and experimental studies indicate that maternal exposure to environmental pollutants impairs the cognitive and motor functions of offspring in humans and laboratory animals. Infant ultrasonic vocalizations (USVs), the communicative behavior of pups toward caregivers, are impaired in rodent models of neurodevelopmental disorders, suggesting a useful method to evaluate the developmental neurotoxicity of environmental pollutants. Therefore, we investigated USVs emitted by mouse pups of dams exposed to 2-chloro-3,7,8-tribromodibenzofuran (TeXDF) and 1,2,3,7,8-pentabromodibenzofuran (PeBDF), which are detected in the actual environment. The USV duration and number in the pups born to dams administered with TeXDF 40 µg/kg body weight (b.w.), but not 8 µg/kg b.w., on gestational day (GD) 12.5, were significantly lower than those in the corresponding pups on postnatal days 3-9. Conversely, there was no statistical change in the USVs emitted by the pups of dams administered with PeBDF 35 or 175 µg/kg b.w. on GD 12.5. To examine whether maternal exposure leads to behavioral impairments in adulthood, we analyzed exploratory behaviors in a novel environment using IntelliCage, a fully automated testing apparatus for group-housed mice. Neither TeXDF nor PeBDF exposure induced significant differences in offspring exploration. Considered together, our findings revealed that TeXDF induces atypical USV emission in infant mice, suggesting the importance of further studies on the risk assessment of mixed brominated/chlorinated dibenzo-p-dioxins and dibenzofurans.

Synthesis and anti-inflammatory activity evaluation of NO-releasing furoxan/1,2,4-triazole hybrid derivatives.

An efficient synthesis method was developed for furoxan/1,2,4-triazole hybrids 5a-k from methyl 5-(halomethyl)-1-aryl-1H-1,2,4-triazole-3-carboxylates 1 through two-steps reaction including hydrolyzation and esterification. All of the furoxan/1,2,4-triazole hybrid derivatives were characterized by spectroscopy. On the other hand, the influence of newly synthesized multi-substituted 1,2,4-triazoles on the exogenous NO release ability, in vitro and in vivo anti-inflammatory activity, and in silico predictions were experimentally evaluated. Based on the exogenous NO release ability study and SAR studies of in vitro anti-inflammatory activity, all of compounds 5a-k exhibited slightly NO release ability and potential anti-inflammatory activity on LPS-induced RAW264.7 cells (IC50 = 5.74-15.3 µM) compared to Celecoxib (IC50 = 16.5 µM) and Indomethacin (IC50 = 56.8 µM). Furthermore, compounds 5a-k were also subjected to in vitro COX-1/COX-2 inhibition assays. Particularly, compound 5f exhibited extraordinary COX-2 inhibition (IC50 = 0.0455 µM) and selectivity (SI = 209). In addition, compound 5f was also examined in vivo pro-inflammatory cytokine productions and gastric safety and possessed the better inhibition of cytokine and safety compared with Indomethacin at the same concentration. Through the molecular modeling and in silico physicochemical and pharmacokinetic properties prediction, compound 5f was stabilized in COX-2 active binding site and possessed the fundamental strong H-bond interaction with Arg499 to form the significant physicochemical and pharmacological properties as a candidate drug. Following the in vitro, in vivo, and in silico study results, compound 5f demonstrated to be a potential anti-inflammatory agent and had comparable effects with Celecoxib.

Rhododendrol, a reductive metabolite of raspberry ketone, suppresses the differentiation of 3T3­L1 cells into adipocytes.

Obesity is a serious medical condition worldwide, and a major risk factor for type 2 diabetes, metabolic syndrome, cancer and cardiovascular disease. In addition to changes in dietary habits and physical activity, consuming supplements to maintain good health and prevent obesity is important in modern society. Raspberry ketone (RK) is a natural phenolic ketone found in the European red raspberry (Rubus idaeus L.) and is hypothesized to prevent obesity when administered orally. The present study found that RK was reduced to rhododendrol (ROH) in human liver microsomes and cytosol. The present study investigated whether the metabolite ROH had anti­adipogenic effects using mouse 3T3­L1 cells. The effects of ROH or RK on lipid accumulation during differentiation of 3T3­L1 pre­adipocyte into adipocyte were determined using Oil Red O staining. CCAAT enhancer­binding protein α (C/EBPα) and peroxisome proliferator­activated receptor γ (PPARγ) mRNA and protein expression were examined using reverse transcription­quantitative PCR and western blotting analysis, respectively. The present study revealed that ROH suppressed lipid accumulation in the cells, similar to RK. In addition, ROH suppressed the mRNA expression levels of C/EBPα and PPARγ in 3T3­L1 adipocytes. Furthermore, ROH suppressed PPARγ protein expression in 3T3­L1 adipocytes. These findings suggested that ROH is an active metabolite with an anti­adipogenic effect, which may contribute to the anti­obesity effect of orally administered RK. The present study indicated that it is important to understand the biological activity of the metabolites of orally administered compounds.

Determination of the relative potencies of brominated dioxins for risk assessment in aquatic environments using the early-life stage of Japanese medaka.

World Health Organization toxic equivalency factors (WHO-TEFs) are recommended for risk management of brominated dioxins in aquatic environments because limited information is available on their toxicity to fish. To validate this approach, we obtained the relative potencies of polybrominated dibenzo-p-dioxins and polybrominated dibenzofurans and mixed-halogenated furans (PXDF, X = Cl/Br) against 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) based on their toxicity to the early-life stage of Japanese medaka (Oryzias latipes). 2,3,7,8-substituted brominated dibenzofurans caused typical dioxin exposure effects, such as blue-sac disease. The TCDD-relative potency factors (REPs) of test substances were calculated based on the concentrations in water and eggs that caused 20% lethality on day 28 post-fertilization, and were in the order of: 2-chloro-3,7,8-tribromodibenzofuran (REPwater 3.3, REPegg 4.6) > 2,3,7,8-tetrabromodibenzofuran (0.85, 0.92) > 2,3,4,7,8-pentabromodibenzofuran (0.053, 0.55) > 1,2,3,7,8-pentabromodibenzofuran (0.0091, 0.19). The transfer rate from water to eggs was lower for pentabrominated furans than tetrabrominated congeners, and was expected to decrease with the log Kow of the test substance. Although the REPegg value can be used to compare the toxicity potential of brominated dioxins, REPwater may be more suitable for environmental risk assessment because the uptake potential of these compounds from water should be considered. This study is the first to report higher toxicity of a PXDF congener compared with TCDD in vivo, further investigations of the toxicity of mixed-halogenated dioxins and environmental behavior are necessary for environmental risk assessment.

Carbonic anhydrase 3 increases during liver adipogenesis even in pre-obesity, and its inhibitors reduce liver adipose accumulation.

The abnormal lipid metabolism in the liver that occurs after high caloric intake is the main cause of nonalcoholic fatty liver disease (NAFLD). Differences between samples from healthy livers and livers from individuals with NAFLD indicate that changes in liver function occur during disease progression. Here, we examined changes in protein expression in a fatty liver model in the early stages of obesity to identify potential alterations in function. The proteins expressed in the liver tissue of pre-obese rats were separated via SDS/PAGE and stained with Coomassie brilliant blue-G250. Peptide mass fingerprinting indicated an increase in the expression of carbonic anhydrase 3 (CA3) relative to controls. Western blotting analysis confirmed the increase in CA3 expression, even in an early fat-accumulation state in which excessive weight gain had not yet occurred. In human hepatoma HepG2 cells, fat accumulation induced with oleic acid also resulted in increased CA3 expression. When the cells were in a state of fat accumulation, treating them with the CA3 inhibitors acetazolamide (ACTZ) or 6-ethoxyzolamide (ETZ) suppressed fat accumulation, but only ETZ somewhat reduced the fat-induced upregulation of CA3 expression. Expression of CA3 was therefore upregulated in response to the consumption of a high-fat diet, even in the absence of an increase in body weight. The suppression of CA3 activity by ACTZ or ETZ reduced fat accumulation in hepatocytes, suggesting that CA3 is involved in the development of fatty liver.

Liver-specific decrease in Tff3 gene expression in infant mice perinatally exposed to 2,3,7,8-tetrabromodibenzofuran or 2,3,7,8-tetrachlorodibenzo-p-dioxin.

Polybrominated dibenzo-p-dioxins and dibenzofurans (PBDD/DFs) are byproducts of brominated flame retardants and can cause adverse health effects. Although exposure to polychlorinated (PC) DD/DFs induces toxic effects, including liver injury and neurobehavioral disorder, little is known about toxicities associated with PBDD/DF exposure. Thus, we examined effects of perinatal exposure to brominated congener on the infant mouse. Gene expression in several organs, such as the liver and brain, was analyzed in mouse offspring born to dams administered 2,3,7,8-tetrabromodibenzofuran (TBDF; 9 or 45 µg/kg body weight) or 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD; 3 µg/kg body weight) on gestational day 12.5. An increase in liver size was observed in TBDF- or TCDD-exposed offspring in infancy. Gene microarray analysis revealed that 163 and 36 genes were markedly upregulated and downregulated, respectively, in the liver of TBDF-exposed mice compared with those in vehicle-treated mice on postnatal day (PND) 5. Significant increases in Cyp1a1, Cyp1a2, Fmo3, and Pnliprp1 and decreases in Tff3, Ocstamp, Kcnk16, and Lgals2 mRNA levels in TBDF-exposed offspring on PNDs 5 and 12 were confirmed by quantitative PCR. In particular, a significant reduction in Tff3 mRNA in the liver, but not in the brain, small intestine, colon, and kidney, was observed in offspring perinatally exposed to TBDF or TCDD. Ultrasonic calls of TBDF- or TCDD-exposed offspring on PNDs 3-5 were impaired. Taken together, perinatal exposure to polyhalogenated dioxin/furan congeners disrupts gene expression patterns in the liver and ultrasonic calling during infancy. These results suggest that liver injury may contribute to neurobehavioral disorder.

Behavioral impairments in infant and adult mouse offspring exposed to 2,3,7,8-tetrabromodibenzofuran in utero and via lactation.

Polybrominated dibenzo-p-dioxins and dibenzofurans (PBDD/DFs) have been unintentionally produced and emitted from the lifecycle of products containing brominated flame retardants, such as polybrominated diphenyl ether, which is suspected to cause developmental neurotoxicity (DNT). Although it is plausible that PBDD/DFs can also induce DNT, information regarding their neurotoxic potential is currently limited. Hence, in the present study, we examined the effects of in utero and lactational exposure to brominated dibenzofurans on infant and adult offspring behavior to understand the mechanism of PBDD/DFs toxicity and detect effective behavioral endpoints in DNT assessment. We analyzed the behavior of mouse offspring born to dams administered 2,3,7,8-tetrabromodibenzofuran (2,3,7,8-TeBDF; dose of 0, 9, or 45 µg/kg) or 2,3,8-tribromodibenzofuran (2,3,8-TrBDF; dose of 0, 75.6, or 378 µg/kg) on gestational day 12.5. In mouse offspring born to dams exposed to 2,3,7,8-TeBDF, the exploratory behavior in a novel environment in adulthood and ultrasonic vocalization (USV) during infancy were significantly reduced. Additionally, AhR-target genes, such as Cyp1a1, were induced in the liver of 2,3,7,8-TeBDF-exposed offspring in a dose-dependent manner. Conversely, no significant changes in the infant and adult behaviors and expression level of AhR-target genes were observed in the 2,3,8-TrBDF-exposed offspring. These results suggest that 2,3,7,8-TeBDF can induce DNT and that the analysis of exploratory behavior in a novel environment and USV may be useful endpoints to assess DNT of dioxin-related substances.

Selective Synthesis and Photoluminescence Study of Pyrazolopyridopyridazine Diones and N-Aminopyrazolopyrrolopyridine Diones.

The newly designed luminol structures of pyrazolopyridopyridazine diones and N-aminopyrazolopyrrolopyridine diones were synthesized from versatile 1,3-diaryfuropyrazolopyridine-6,8-diones, 1,3-diarylpyrazolopyrrolopyridine-6,8-diones, or 1,3-diaryl-7-methylpyrazolopyrrolopyridine-6,8-diones with hydrazine monohydrate. Photoluminescent and solvatofluorism properties containing UV-Vis absorption, emission spectra, and quantum yield (Φf) study of pyrazolopyridopyridazine diones and N-aminopyrazolopyrrolopyridine diones were also studied. Generally, most of pyrazolopyrrolopyridine-6,8-diones 6 exhibited the significant fluorescence intensity and the substituent effect when compared with N-aminopyrazolopyrrolopyridine diones, particularly for 6c and 6j with a m-chloro group. Additionally, the fluorescence intensity of 6j was significantly promoted due to the suitable conjugation conformation. Based on the quantum yield (Φf) study, the value of compound 6j (0.140) with planar structural skeletal was similar to that of standard luminol (1, 0.175).

Activation of PXR, CAR and PPARα by pyrethroid pesticides and the effect of metabolism by rat liver microsomes.

In this study, we used reporter gene assays in COS-1 cells to examine the activation of rat pregnane X receptor (PXR), rat constitutive androstane receptor (CAR) and rat peroxisome-proliferator activated receptor (PPAR)α by pyrethroid pesticides, and to understand the effects of metabolic modification on their activities. All eight pyrethroids tested in this study showed rat PXR agonistic activity; deltamethrin was the most potent, followed by cis-permethrin and cypermethrin. However, when the pyrethroids were incubated with rat liver microsomes, their rat PXR activities were decreased to various extents. Cis- and trans-permethrin showed weak rat CAR agonistic activity, while the other pyrethroids were inactive. However, fenvalerate showed dose-dependent inverse agonistic activity toward rat CAR, and this activity was reduced after metabolism. None of the pyrethroids showed rat PPARα agonistic activity, but a metabolite of cis-/trans-permethrin and phenothrin, 3-phenoxybenzoic acid, activated rat PPARα. Since PXR, CAR and PPARα regulate various xenobiotic/endobiotic-metabolizing enzymes, activation of these receptors by pyrethroids may result in endocrine disruption due to changes of hormone-metabolizing activities.

Comparative study of the effect of 17 parabens on PXR-, CAR- and PPARα-mediated transcriptional activation.

Parabens are widely used as preservatives in personal care products, medicines and foods, resulting in substantial human exposures, even though some harmful effects, such as endocrine-disrupting activity, have been reported. Pregnane X receptor (PXR), constitutive androstane receptor (CAR) and peroxisome proliferator-activated receptor α (PPARα), which are members of the nuclear receptor superfamily, regulate the metabolism of endogenous substrates including hormones. Therefore, we hypothesized that parabens may alter hormone-metabolizing activities by acting on these receptors, and such changes could contribute to the endocrine-disrupting activity. To test this idea, we systematically examined the effects of 17 parabens on these receptors using reporter gene assays. Nine parabens significantly activated human and rat PXR. Parabens with C2-C5 (linear and branched) side chains were most active. Butylparaben and isobutylparaben also significantly activated rat CAR. We found that long-side-chain (C7-C12) parabens showed up to 2-fold activation of PPARα at 10 µM. Furthermore, pentylparaben and hexylparaben showed rat PXR antagonistic activity and rat CAR inverse agonistic activity. The activity of butylparaben towards PXR and CAR was lost after carboxylesterase-mediated metabolism. These findings confirm that parabens influence the activities of PXR, CAR and PPARα, and thus have the potential to contribute to endocrine disruption by altering hormone metabolism.

Profiling of bisphenol A and eight its analogues on transcriptional activity via human nuclear receptors.

Several bisphenol A (BPA) analogues have been detected in environmental samples, foodstuffs, and/or human biological samples, and there is concern regarding their potential endocrine-disrupting effects. In this study, we characterized the agonistic and/or antagonistic activities of BPA and eight its analogues against human estrogen receptors (ERα/ß), androgen receptor (AR), glucocorticoid receptor (GR), pregnane X receptor (PXR), and constitutive androstane receptor (CAR). All the test compounds, except for bisphenol P (BPP), showed both ERα and ERß agonistic activities, with bisphenol AF (BPAF) being the most potent. On the other hand, BPAF and BPP showed ERα and ERß antagonistic activities. Interestingly, their ER activities demonstrated a preference toward ERß. All the test compounds, except for bisphenol S, showed AR antagonistic activities, with bisphenol E being the most potent. Weak GR antagonistic activities were also found in BPA and five its analogues. PXR agonistic activity was observed in the six compounds, with bisphenol Z being the most potent. Results of the CAR assay revealed that BPA and five its analogues acted as CAR inverse agonists. Taken together, these results suggested that BPA analogues demonstrate multiple effects via human nuclear receptors in a similar manner to BPA, and several analogues might have more potent endocrine-disrupting activity than does BPA.

Identification of hepatic thyroid hormone-responsive genes in neonatal rats: Potential targets for thyroid hormone-disrupting chemicals.

There have been many concerns about the possible adverse effects of thyroid hormone-disrupting chemicals in the environment. Because thyroid hormones are essential for regulating the growth and differentiation of many tissues, disruption of thyroid hormones during the neonatal period of an organism might lead to permanent effects on that organism. We postulated that there are target genes that are sensitive to thyroid hormones particularly during the neonatal period and that would thus be susceptible to thyroid hormone-disrupting chemicals. Global gene expression analysis was used to identify these genes in the liver of rat neonates. The changes in hepatic gene expression were examined 24 h after administering 1.0, 10, and 100 ng/g body weight (bw) triiodothyronine (T3) to male rats on postnatal day 3. Thirteen upregulated and four downregulated genes were identified in the neonatal liver. Among these, Pdp2 and Slc25a25 were found to be upregulated and more sensitive to T3 than the others, whereas Cyp7b1 and Hdc were found to be downregulated even at the lowest dose of 1.0 ng/g bw T3. Interestingly, when the responses of gene expression to T3 were examined in adult rats (8-week old), one-third of them did not respond to T3. The environmental chemicals with thyroid hormone-like activity, hydroxylated polybrominated diphenyl ethers, were then administered to neonatal rats to examine the effects on expression of the identified genes. The results showed that these chemicals were indeed capable of changing the expression of Slc25a25 and Hdc. Our results demonstrated a series of hepatic T3-responsive genes that are more sensitive to hormones during the neonatal period than during adulthood. These genes might be the potential targets of thyroid hormone-disrupting chemicals in newborns.

One-Flask Synthesis of Pyrazolo[3,4-d]pyrimidines from 5-Aminopyrazoles and Mechanistic Study.

A novel one-flask synthetic method was developed in which 5-aminopyrazoles were reacted with N,N-substituted amides in the presence of PBr3. Hexamethyldisilazane was then added to perform heterocyclization to produce the corresponding pyrazolo[3,4-d]pyrimidines in suitable yields. These one-flask reactions thus involved Vilsmeier amidination, imination reactions, and the sequential intermolecular heterocyclization. To study the reaction mechanism, a series of 4-formyl-1,3-diphenyl-1H-pyrazol-5-yl-N,N-disubstituted formamidines, which were conceived as the chemical equivalent of 4-(iminomethyl)-1,3-diphenyl-1H-pyrazol-5-yl-formamidine, were prepared and successfully converted into pyrazolo[3,4-d]pyrimidines. The experiments demonstrated that the reaction intermediates were the chemical equivalents of 4-(iminomethyl)-1,3-diphenyl-1H-pyrazol-5-yl)formamidines. The rate of the reaction could be described as being proportional to the reactivity of amine reactants during intermolecular heterocyclization, especially when hexamethyldisilazane was used.

Protective Effects of Tormentic Acid, a Major Component of Suspension Cultures of Eriobotrya japonica Cells, on Acetaminophen-Induced Hepatotoxicity in Mice.

An acetaminophen (APAP) overdose can cause hepatotoxicity and lead to fatal liver damage. The hepatoprotective effects of tormentic acid (TA) on acetaminophen (APAP)-induced liver damage were investigated in mice. TA was intraperitoneally (i.p.) administered for six days prior to APAP administration. Pretreatment with TA prevented the elevation of serum aspartate aminotransferase (AST), alanine aminotransferase (ALT), total bilirubin (T-Bil), total cholesterol (TC), triacylglycerol (TG), and liver lipid peroxide levels in APAP-treated mice and markedly reduced APAP-induced histological alterations in liver tissues. Additionally, TA attenuated the APAP-induced production of nitric oxide (NO), reactive oxygen species (ROS), tumor necrosis factor-alpha (TNF-α), interleukin-1beta (IL-1ß), and IL-6. Furthermore, the Western blot analysis showed that TA blocked the protein expression of inducible NO synthase (iNOS) and cyclooxygenase-2 (COX-2), as well as the inhibition of nuclear factor-kappa B (NF-κB) and mitogen-activated protein kinases (MAPKs) activation in APAP-injured liver tissues. TA also retained the superoxidase dismutase (SOD), glutathione peroxidase (GPx), and catalase (CAT) in the liver. These results suggest that the hepatoprotective effects of TA may be related to its anti-inflammatory effect by decreasing thiobarbituric acid reactive substances (TBARS), iNOS, COX-2, TNF-α, IL-1ß, and IL-6, and inhibiting NF-κB and MAPK activation. Antioxidative properties were also observed, as shown by heme oxygenase-1 (HO-1) induction in the liver, and decreases in lipid peroxides and ROS. Therefore, TA may be a potential therapeutic candidate for the prevention of APAP-induced liver injury by inhibiting oxidative stress and inflammation.

Construction of the Database of Rat Repeated-dose Toxicity Tests of Pesticides for the Toxicological Characterization of Hepatocyte Hypertrophy.

Liver and hepatocyte hypertrophy can be induced by exposure to chemical compounds, but the mechanisms and toxicological characteristics of these phenomena have not yet been investigated extensively. In particular, it remains unclear whether the hepatocyte hypertrophy induced by chemical compounds should be judged as an adaptive response or an adverse effect. Thus, understanding of the toxicological characteristics of hepatocyte hypertrophy is of great importance to the safety evaluation of pesticides and other chemical compounds. To this end, we have constructed a database of potentially toxic pesticides. Using risk assessment reports of pesticides that are publicly available from the Food Safety Commission of Japan, we extracted all observations/findings that were based on 90-day subacute toxicity tests and 2-year chronic toxicity and carcinogenicity tests in rats. Analysis of the database revealed that hepatocyte hypertrophy was observed for 37-47% of the pesticides investigated (varying depending on sex and testing period), and that centrilobular hepatocyte hypertrophy was the most frequent among the various types of hepatocyte hypertrophy in both the 90-day and 2-year studies. The database constructed in this study enables us to investigate the relationships between hepatocyte hypertrophy and other toxicological observations/findings, and thus will be useful for characterizing hepatocyte hypertrophy.

Altered expression of the Olr59, Ethe1, and Slc10a2 genes in the liver of F344 rats by neonatal thyroid hormone disruption.

Many concerns have been expressed regarding the possible adverse effects of thyroid hormone-disrupting chemicals in the environment. The disruption of thyroid hormones in the neonatal period may lead to permanent effects on thyroid hormone homeostasis as well as related developmental disorders, as thyroid hormones are essential for regulating the growth and differentiation of many tissues. To understand the long-term alteration in gene expressions by neonatal administration of thyroid hormone-like chemicals in general, we identified genes whose expression was altered in the liver, an important component of the thyroid hormone axis, by neonatal exposure to triiodothyronine (T3). T3 was administered to male F344 rats on postnatal days 1, 3, and 5 (week 0). At 8 weeks of age, cDNA microarray analysis was used to identify hepatic genes whose expression was altered by neonatal exposure to T3. Among the up-regulated genes that were identified, the expression of Olr59, Ethe1, and Slc10a2 increased specifically in rats neonatally exposed to T3. Interestingly, altered hepatic expression of these genes indeed increased when a hydroxylated polybrominated diphenyl ether (PBDE), OH-BDE42, which is capable of binding to the TR, was given neonatally. Our data demonstrated that neonatal exposure to thyroid hormones could affect the long-term expression of the genes, which could be useful markers for neonatal effects by thyroid hormone-disrupting chemicals. Copyright © 2017 John Wiley & Sons, Ltd.

Comparative study of hydrolytic metabolism of dimethyl phthalate, dibutyl phthalate and di(2-ethylhexyl) phthalate by microsomes of various rat tissues.

Phthalates are used in food packaging, and are transferred to foods as contaminants. In this study, we examined the hydrolytic metabolism of dimethyl phthalate (DMP), dibutyl phthalate (DBP) and di(2-ethylhexyl) phthalate (DEHP) by rat tissue microsomes. We found that carboxylesterase and lipase contribute differently to these activities. When DMP, DBP and DEHP were incubated with rat liver microsomes, DBP was most effectively hydrolyzed to the phthalate monoester, followed by DMP, and the activity toward DEHP was marginal. In contrast, small-intestinal microsomes exhibited relatively higher activity toward long-side-chain phthalates. Pancreatic microsomes showed high activity toward DEHP and DBP. Liver microsomal hydrolase activity toward DMP was markedly inhibited by bis(4-nitrophenyl)phosphate, and could be extracted with Triton X-100. The activity toward DBP and DEHP was partly inhibited by carboxylesterase inhibitor, and was partly solubilized with Triton X-100. Ces1e, Ces1d and Ces1f expressed in COS cells exhibited the highest hydrolase activity toward DBP, showing a similar pattern to that of liver microsomes. Ces1e showed activity towards DMP and DEHP. Pancreatic lipase also hydrolyzed DBP and DEHP. Thus, carboxylesterase and lipase contribute differently to phthalate hydrolysis: short-side-chain phthalates are mainly hydrolyzed by carboxylesterase and long-side-chain phthalates are mainly hydrolyzed by lipase.

Variability of Zaleplon 5-Oxidase Activity in Mice and Humans, and Inhibition by Raloxifene.

BACKGROUND: Zaleplon (ZAL) is a sedative-hypnotic agent, which is mainly metabolized to inactive 5-oxidized zaleplon (5-oxo-ZAL) and N-des-ethylated ZAL (des-ethyl-ZAL) in mice and humans. The former reaction is considered to be catalyzed by aldehyde oxidase present in liver cytosol. METHODS: Here, we examined sex and strain differences of ZAL metabolism to 5-oxo-ZAL among four strains of mice, as well as the inter-individual variation in humans, in order to evaluate the variability of 5-oxo-ZAL-forming activity and its relationship with aldehyde oxidase activity. In mice, the activity in C57BL/6J strain was the highest, followed by C3H/He and BALB/c. The activity in DBA/2J was the lowest, being 2.3-fold lower than that of C57BL/6J mice. The activity of male mice was higher than that of female mice. Large inter-individual variations were observed among humans, with a range of 10- fold. Raloxifene, an inhibitor of aldehyde oxidase, markedly decreased the formation of 5-oxo-ZAL by liver cytosol of mice and humans. Further, the plasma level of 5-oxo-ZAL in mice was decreased when raloxifene was co-administered with ZAL. RESULTS: Our results indicate that the formation of 5-oxo-ZAL from ZAL is mainly catalyzed by aldehyde oxidase in mice and humans, and the variability of 5-oxo-ZAL formation is due primarily to differences of aldehyde oxidase activity. CONCLUSION: High inter-individual variability of ZAL 5-oxidase activity and potential for interaction of ZAL with other medicines that are inhibitors of aldehyde oxidase should be taken into consideration in clinical usage of ZAL.