Human monocytic cell line THP-1 cells are used as an indicator for in vitro skin sensitization testing. Although p38 mitogen-activated protein kinases (MAPKs) and intracellular redox imbalance play crucial roles in the activation of THP-1 by skin sensitizers, the trigger of cell activation has not been identified. Therefore, we examined whether haptens induce THP-1 maturation directly or indirectly. 2,4-Dinitrochlorobenzene (DNCB), but not dinitrophenol (DNP)-conjugated bovine serum albumin or DNP-conjugated fetal bovine serum, induced CD86 expression. DNCB and nickel sulfate (NiSO4) also induced related changes of cell-surface thiols and phosphorylation of p38 MAPK. However, DNCB is membrane-permeable, and so its direct effect may not be confined to cell membrane proteins. Next, we found that CD86 expression and macrophage inflammatory protein-1β (MIP-1β) production were augmented by the membrane-impermeable thiol blocker 5,5'-dithiobis(2-nitrobenzoic acid) (DTNB), and these changes were suppressed by an inhibitor of the p38 MAPK pathway, SB203580. Finally, we confirmed that endocytotic activity for bovine serum albumin (BSA) Alexa Fluor 488 conjugate did not affect cell-surface thiols on THP-1 cells. Thus, our data indicate that the changes of cell-surface thiols are one of the triggers of maturation, and play a key role in activation of THP-1 cells by haptens.
Prolactin has a wide variety of biological effects. Consequences of hyperprolactinemia on islet B cell proliferation as well as general toxicological parameters were here examined using anterior pituitary-grafted rats. Three or six anterior pituitary glands were implanted under single renal capsules of F344 male rats and left there for 13 weeks afterward. Clinical observation along with measurement of body weight and food consumption was conducted during the observation period, and subsequently hematology, blood biochemistry, gross pathology, organ weights and histopathology were examined. In addition, the proliferation rate of islet B cells was measured by a 5-bromo-2'-deoxy-uridine (BrdU) labeling technique. Serum prolactin concentrations at week 13 were 36, 70, 75 and 105 ng/ml in the sham-operated, 3-pituitary-grafted groups from male or female donors, and 6-pituitary-grafted group from male donors, respectively. Higher cholinesterase and total cholesterol values, lower trigriceride and leutenizing hormones (LH) values, and higher adrenal weights compared to those in the sham-operated group were apparent in the 3- and/or 6-pituitary-grafted groups. Also, the study revealed that mammary gland structure was transformed with change of differentiation from a male to a female acinar pattern. Furthermore a specific increase of islet cell proliferation rate was found, positively correlated with serum prolactin concentration. These findings suggest that elevation of serum prolactin level over 13 weeks induces islet cell proliferation and changes in toxicological parameters, including cholinesterase activity, elements of lipid metabolism and histopathology/morphology of the adrenals and mammary glands in male rats.
The present study was conducted to assess involvement of oxidative stress in lung adeno-carcinogenesis, using mice deficient in the 8-hydroxyguanine DNA glycosylase 1 (Ogg1) gene encoding an enzyme that repairs an oxidative DNA injury 8-oxoguanine (8-oxoG). Furthermore, for comparison with the human case, mutations of mouse epidermal growth factor receptor (Egfr) and K-ras genes were examined. The homo- and heterozygously Ogg1 gene-deficient and wild-type mice (C57BL6/J origin), 6 weeks old, were administered 4-(N-hydroxymethylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK) by continuous subcutaneous infusion using an osmotic pump at a total dose of 6 mg/mouse for 1 week, then treated with one of 4 antioxidants (phenyl N-tert-butyl nitrone 0.13% in drinking water, resveratrol 20 ppm in diet, lactoferrin 2% in diet and bilberry powder 2% in diet) or no supplement for 33 weeks. Development of lung adenomas and preneoplastic atypical hypreplasias was significantly enhanced by the homo- and heterozygous Ogg1 gene deficiency only in female mice with intralesion accumulation of 8-oxoG. All antioxidants tended to inhibit enhanced adeno-carcinogenesis. The Egfr and K-ras gene mutations were detected at sites also found in human lung cancers with low incidences, while the Egfr gene mutation was detected for the first time in chemical lung carcinogenesis of animals. It is indicated that the Ogg1 gene deficiency enhances lung adeno-carcinogenesis in mice by virtue of accelerated oxidative stress. The presently utilized Ogg1 gene-deficient mice model may be useful to draw mechanism-based strategies to control human lung adenocarcinomas, especially in women.
Carbon tetrachloride (CCl4) is well known to induce hepatotoxicity after being metabolized to trichloromethyl free radical (·CCl3) by CYP2E1. In the present study, the hepatotoxicity induced by a single oral dose (2,000 mg/kg) of CCl4 was compared between pregnant (gestation days (GD) 13 and 19) or postpartum (postpartum days (PPD) 1, 13 and 27) and non-pregnant rats. Hepatotoxicity in CCl4-treated pregnant rats evaluated by blood chemistry (alanine aminotransferase (ALT), aspartate aminotransferase (AST) and lactate dehydrogenase (LDH) activities) and histopathological finding (area of damaged hepatocytes) was minimal on GD19, being weaker than that in non-pregnant rats. CYP2E1 expression in non-treated pregnant rats decreased as pregnancy progressed and reached minimum level on GD19. Thus, the degree of CCl4-induced hepatotoxicity roughly corresponded to CYP2E1 levels during pregnancy. After delivery, hepatotoxicity in CCl4-treated lactating rats was maximal on PPD13, being stronger than that in non-pregnant rats, and then it decreased slightly on PPD27. The CYP2E1 level in the non-treated lactating rats tended to increase but remained at lower levels until PPD13 compared with that in non-pregnant rats. Thus, the degree of CCl4-induced hepatotoxicity did not correspond to CYP2E1 levels during lactation. This suggests that during lactation, there may be certain factors other than CYP2E1 expression responsible for the degree of CCl4-induced hepatotoxicity.
The assessment of drug effects on attention is important in non-clinical pharmacology, for both evaluation of safety and therapeutic efficacy of medicinal products. In the present study, we have developed a two-lever choice behavioral test to assess drug effects on attentional performance in monkeys. In each trial of this experiment, one of two lamps in front of a monkey was randomly illuminated for a brief period of time and the monkey was required to press a lever beneath the lamp 30 times to obtain a food reward. The percentage of correct responses, response latency of correct choice responses and response speed were measured. Using this test, we examined the effects of three sedative drugs, diazepam (0.25, 1 and 4 mg/kg, i.g.), ethanol (0.5, 1 and 2 g/kg, i.g.), and pentobarbital (0.25, 1 and 4 mg/kg, i.v.). Diazepam and pentobarbital lengthened response latency without significantly affecting the percentage of correct responses, response and response speed, suggesting selective disruptive effects on attentional performance. In contrast, ethanol at the high dose tested caused deterioration in all three measurements, which is thought to reflect a general sedative effect including motor impairment as reflected by lengthening response speed. It is suggested that the present behavioral test method could detect drug effects on attentional performance in monkeys and could be a useful tool for safety assessment in drug development.
Se-Methylselenocysteine (MeSeCys) is not only a selenium (Se) supplement but also a more promising precursor of an anti-tumor drug containing Se than selenomethionine, which is currently used as Se supplement. In this study, the metabolism of MeSeCys labeled with an Se isotope, 82Se, in rats depleted of endogenous natural abundance isotopes with another Se isotope, 78Se, was traced for 21 days when MeSeCys was continuously and perorally ingested at a supplemental dose. The tracer experiment was performed with our improved method that utilized an inductively coupled plasma-deuterium reaction-mass spectrometer. The substitution of endogenous Se with a single isotope, 78Se, facilitated the detection of exogenous labeled Se. Exogenous Se in the form of MeSeCys preferably accumulated and/or assimilated in the liver, kidneys and testes with long-term ingestion of MeSeCys and was utilized for the synthesis of selenoproteins, i.e., extracellular and cellular glutathione peroxidases and selenoprotein P. Meanwhile, intact MeSeCys was not excreted into urine although trimethylselenonium was detected in addition to selenosugar. The results suggest that MeSeCys was transformed into selenide via methylselenol by β-lyase. Consequently, it is surmised that MeSeCys is a precursor of methylselenol under long-term ingestion.
Escherichia coli WP2uvrA/pKM101 strain obtained from a different supplier had a lower response to quinolone antibacterial agents (quinolones) when compared to an ordinary responsive strain. The present study was designed to examine the mechanism of lower susceptibility of the new strain to quinolones. A reverse mutation assay showed the new strain was low responsive to six quinolones compared to a responsive strain, while there was no difference in sensitivity to antibacterial activity of quinolones or the mutagenic activity of the positive control compounds in both strains. The sequence of mucA and B genes, which involve chemical and ultraviolet (UV) -induced mutagenesis through error-prone repair, and the total length of plasmid pKM101 in both strains were identical. Furthermore, transformed WP2uvrA/pKM101 strains, which were made by separately electroporating pKM101 extracted from these two strains into WP2uvrA, showed almost the same responses to the mugatenic- and antibacterial- activity of quinolones as the original responsive strain. The two original strains and the recipient WP2uvrA were proven to have proper genetic characteristics. It was demonstrated that the lower susceptibility of the new WP2uvrA/pKM101 strain to the mutagenesis of quinolones was not due to any changes in either plasmid pKM101 or the characteristics of the host detected by a routine check (tryptophan requirements, sensitivity to UV light and cell membrane permeability) of their genetic characteristics.
Wistar Hannover Global Alliance for Laboratory Animal Standardization (WH GALAS) rats have been distributed for international standardization of preclinical and toxicological research. Han/Wistar (Kuopio) rats are exceptionally resistant to acute toxicities caused by 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) and mediated by the aryl hydrocarbon receptor (AhR), and they have a mutated AhR, named AhRhw/hw. We found that the WH GALAS rat has either of the three AhR allele, AhRwt/wt, AhRwt/hw and AhRhw/hw. We administered TCDD (0, 5 and 10 µg/kg) to Long-Evans (L-E) rats having AhRwt/wt and two WH GALAS rat strains having either AhRwt/wt or AhRhw/hw, and examined the weights of their body, liver and thymus 168 hr post-administration. WH GALAS AhRhw/hw strain was more resistant to TCDD-induced effects on thymus weight than L-E and WH GALAS AhRwt/wt strains. In order to study differences in susceptibility of thymic atrophy among the strains, we examined CYP1A1 mRNA and AhR protein levels between L-E and WH GALAS strains. However, no significant difference was observed in the amount of AhR protein or CYP1A1 mRNA in the thymus. Next, we carried out in vitro assays to examine the transactivation activities of AhR variants and found that the AhR deletion variant (AhRdv) transcribed from AhRhw/hw significantly enhanced transactivation activity of the synthesized xenobiotic response element. All AhR variants similarly suppressed the growth of Jurkat T cells upon TCDD exposure. This study suggests that WH GALAS rat having different AhR alleles is an interesting experimental animal model but should be utilized with caution for preclinical research on chemicals having AhR agonistic activities.
In order to obtain basic risk assessment data on human health exposure to volatile organic compound (VOC) vapor by inhalation, a whole-body inhalation exposure system which allows blood collection during the exposure period was designed. The system was tested using chloroform as a model VOC. Chloroform vapor, sampled from the supply-header, animal-chambers and exhaust-header, remained constant in this system with variations in its concentration being less than 2%; flow rate of the vapor through the system was also constant. Rats were exposed to chloroform vapor and blood collected from the tail during exposure to the chloroform vapor. The chloroform concentration in the blood increased during the initial 60 min of exposure, and afterwards its concentration remained at about 2 µg/ml from 60 to 360 min. In conclusion, our design allows blood to be collected from individual rats during exposure by inhalation to test VOCs and changes in the blood concentration of the VOC during exposure to be assessed.
Pharmaceutical and personal care products (PPCPs) enter aquatic environments via sewage treatment facilities and their potentially toxic effects on biota, particularly aquatic organisms, are of considerable concern. In this study, we investigated the acute toxicity of selected PPCPs on a freshwater crustacean (Thamnocephalus platyurus) and a fish species (Oryzias latipes). The 24-hr median lethal concentration (LC50) values of ibuprofen, mefenamic acid, indometacin, carbamazepine, propranolol, ifenprodil, clarithromycin and triclosan for T. platyurus were estimated to be 19.59, 3.95, 16.14, > 100, 10.31, 4.43, 94.23 and 0.47 mg/l respectively. Conversely, the 96-hr LC50 values for these PPCPs were estimated at > 100, 8.04, 81.92, 45.87, 11.40, 8.71, > 100 and 0.60 mg/l for O. latipes, respectively. The toxic sensitivity of T. platyurus to these PPCPs, except for carbamazepine, was therefore higher than for O. latipes. No acute toxicity effects were associated with PPCPs, such as atenolol, disopyramide, famotidine, fluconazole, erythromycin and levofloxacin, in the two aquatic organisms at the concentrations tested in this study (> 100 mg/l). These findings may help us to understand the potential biological effects and risks associated with PPCP exposure in aquatic organisms. Further long-term studies are required to fully assess the growth and reproduction of these compounds on aquatic biota.
Oral administration of 2,4-dichloro-1-nitrobenzene (2,4-DCNB) causes kidney tumors in the rat. The objective of the present study was to identify the chemical structure of 2,4-DCNB metabolites in urine. Urine from 2,4-DCNB fed rats was more yellow than urine from control rats, exhibiting a broad UV-spectrum around a peak wavelength of 360 nm; the control urine did not have an absorbance. The yellow component was extracted and analyzed. The optical properties of the yellow component were the same as the N-acetylcysteine conjugate of 1,4-dichloro-2-nitrobenzene (1,4-DCNB): 1,4-DCNB is secreted in urine as an N-acetylcysteine conjugate. LC-MS/MS analyses of this yellow component demonstrated its chemical structure to be the N-acetylcysteine conjugate of 2,4-DCNB. Nuclear overhauser effect and LC-MS/MS analyses revealed the structural isomer of this 2,4-DCNB metabolite as N-acetyl-S-(5-chloro-2-nitrophenyl)-L-cysteine. We also discuss the possibility that the N-acetylcysteine conjugate identified in this study plays a role as a proximate carcinogen in the formation of kidney tumors.
It is well known that heavy oil pollution results in various negative impacts on the marine environment. Although there is a low possibility of direct exposure to heavy oil, the chemical substances contained in heavy oil may be released into the environment and accumulated by marine organisms which in turn can be taken by humans via the food chain. In this study, we examined the biological risk of heavy oil extract using the common mouse, whose genetic backgrounds and immune system are well known and relatively homologous to humans. Water-soluble fraction (WSF) was extracted from heavy oil with water and the extract orally administrated to female or male mice for 7 days. In the WSF administrated group, cystoma-like formation was observed in the ovary in approximately 80% of female mice. On the other hand, we found that the prostate gland size in male mice was markedly reduced in comparison with male control mice. Continuous administration of WSF for 28 days resulted in continued hypertrophy of the cystoma around the ovary and atrophy in the prostate gland. In addition, it was revealed that chemical substances within WSF have estrogenic activity. A major component of heavy oil, polycyclic aromatic hydrocarbons (PAHs), is known to present estrogenic activity. It is likely that the cystoma-like formation in female mice and atrophy of prostate gland in male resulted of estrogenic substances present in the WSF which might be the PAHs.