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Archives of Toxicology (v.80, #5)

Ochratoxin A secretion by ATP-dependent membrane transporters in Caco-2 cells by Jan Schrickx; Yuri Lektarau; J. Fink-Gremmels (pp. 243-249).

The ATP-dependent membrane transporters, P-gp, MRP2 and BCRP, localized in the luminal membranes of the intestines, liver and kidney, counteract absorption and increase excretion of xenobiotics and drugs. Previously, it has been suggested that the mycotoxin ochratoxin A (OTA) is a substrate for ATP-dependent transporters, and hence the absorption and secretion of OTA in the Caco-2 cell model was investigated. To this end, Caco-2 cells were cultured as confluent monolayers in bicameral inserts and the transepithelial transport of the mycotoxin was assessed. Caco-2 cells secreted OTA to the luminal side in a concentration-dependent manner. This secretory permeability was higher than the absorptive permeability, while the absorptive permeability remained constant for all OTA concentrations tested. The secretion decreased and absorption increased in the presence of the MRP-inhibitor MK571, the P-gp and BCRP inhibitor GF120918, and the BCRP-inhibitor Ko143, suggesting that the secretion of OTA is mediated by MRP2 and BCRP. Cyclosporine A also decreased the secretory permeability, but did not affect absorptive permeability, while PSC833 did neither change absorption nor secretion of OTA. Hence it can be suggested that OTA is a substrate for MRP2 as well as BCRP. These findings are of interest in evaluating mycotoxin absorption after oral ingestion, tissue distribution and particularly excretion pathways, including renal, biliary and mammary gland excretion.

Keywords: Ochratoxin A; Transport; Caco-2; MRP2; BCRP

Expression of the theta class GST isozyme, Yd_fYd_f, in low GST dogs by Toshiyuki Watanabe; Yoshihiko Ohashi; Toshiyuki Kosaka; Shingo Arakawa; Yukari Shibaya; Takashi Yamoto; Sunao Manabe; Wataru Takasaki (pp. 250-257).

We have reported the existence of low glutathione S-transferase (GST) dogs whose GST activity to 1,2-dichloro-4-nitrobenzene (DCNB) as a substrate (GST-D activity) is quite low, and have also reported significant individual differences in dog liver GST-D activity. The dogs were classified as “low”, “middle”, or “high” GST dogs based on their GST-D activity. In the present study, in order to investigate the causes of quite low GST-D activity in low GST dogs and the individual differences in dog GST-D activity, glutathione (GSH) conjugation of DCNB was kinetically analyzed. Moreover, liver cytosolic proteins whose expression levels were significantly lower in low GST dogs than in high GST dogs were identified by two-dimensional difference gel electrophoresis (2D-DIGE) and LC tandem mass spectrometry (LC/MS/MS). Interestingly, Vmax values for this reaction well reflected their GST-D activities in all groups, i.e. they were 3.8, 80.6, and 169.2 nmol/min/mg protein in the low, middle, and high GST dogs, respectively. However, Km values were almost the same (260.0–283.7 μM) among these groups. These suggest that GSH conjugation of DCNB should be catalyzed by the same enzyme in all the dogs, and individual differences in the GST-D activity should be the result of individual differences in the expression level of the GST isozyme, which catalyzes conjugation of DCNB. In 2D-DIGE, the expression levels of the two protein spots were significantly lower in the low GST dogs than in the high GST dogs. Positive good correlation (r>0.800) was observed between GST-D activity and expression levels of these two protein spots. Moreover, expression levels were quite low in low GST dogs. These two proteins were both identified as the theta class GST isozyme, Yd_fYd_f, which specifically catalyzes GSH conjugation of DCNB in dog livers. In the present study, we present two novel findings based on an enzyme kinetic study and protein-expression analysis: (1) GSTYd_fYd_f is expressed at quite a low level in the liver of low GST dogs, and (2) individual differences in dog liver GST-D activity would be due to individual differences in the expression level of GSTYd_fYd_f. Considering these findings, low GST dogs might have high susceptibility, including an unexpected toxicity at abnormal exposure to chemicals metabolized by GSTYd_fYd_f.

Keywords: Glutathione S-transferase; Low GST dog; GSTYd_fYd_f ; Deficiency; 2D-DIGE

Induction of CYP1A1 in rat lung cells following in vivo and in vitro exposure to quartz by Andrea Becker; Catrin Albrecht; Ad M. Knaapen; Roel P. F. Schins; Doris Höhr; Kirstin Ledermann; Paul J. A. Borm (pp. 258-268).

Respirable quartz has been classified as a human lung carcinogen, but the mechanism by which quartz exposure leads to lung cancer has not been clarified. Consistently higher risks of lung cancer are reported in smokers with quartz exposure and we therefore hypothesised that quartz exposure may alter the expression of enzyme systems involved in activation/detoxification of pre-carcinogens in cigarette smoke. More specifically we studied cytochrome P4501A1 (CYP1A1) expression using reverse transcriptase polymerase chain reaction and immunohistochemistry (IHC) upon in vitro and in vivo quartz exposure. In vitro incubation of rat lung epithelial cells with DQ12 quartz for 24 h showed a dose-dependent induction of CYP1A1-mRNA. On the other hand, CYP1A1 message was not increased in lung epithelial cells isolated from rats at 3, 28 or 90 days after intratracheal instillation of 2 mg DQ12. Following IHC for CYP1A1 protein in rat lung sections from later time-points (180 and 360 days), we observed an increase in the number of CYP1A1 positive cells. After in vivo quartz exposure, protein expression of the Aryl hydrocarbon receptor (AhR) was increased and nuclear translocation of AhR was observed at the same time-points. In conclusion, our findings demonstrate an effect of quartz exposure on chronic CYP1A1 expression in vivo, whereas the in vitro models show an immediate upregulation. We suggest that this upregulation of CYP1A1 may act as a co-carcinogenic pathway in quartz exposed workers by activation of pre-carcinogens such as those present in cigarette smoke.

Keywords: Quartz; CYP1A1; AhR; Lung; Epithelial cells

Toxic serum factor long after single exposure to organophosphate; a new approach for biomonitoring by Tamar Berman-Shlomovich; Uri Wormser; Berta Brodsky (pp. 269-274).

One of the major limitations of current methods of biological detection of exposure to hazardous environmental agents is their inability to detect long-term exposures. In the current study we examined the potential of a new bioassay based on the hypothesis that serum of exposed individuals contains a toxic factor(s) produced by an affected cell/tissue. The procedure included exposure of neuronal PC12 cell cultures to sera of rats treated once with the organophosphate chlorpyrifos. Samples taken 4 weeks after chlorpyrifos exposure reduced nerve growth factor (NGF)-induced neurite outgrowth by 40%. This effect lasted 6 weeks after treatment, whereas motor activity and cholinesterase activity returned to normal levels within 1 week. These results demonstrate the potential of the proposed method to detect environmental exposures long after they have occurred.

Keywords: Organophosphate; Biomonitoring; Chlorpyrifos

The role of toll-like receptor 4 in airway inflammation induced by diesel exhaust particles by Ken-ichiro Inoue; Hirohisa Takano; Rie Yanagisawa; Seishiro Hirano; Takamichi Ichinose; Akinori Shimada; Toshikazu Yoshikawa (pp. 275-279).

Although several studies have demonstrated that airway exposure to diesel exhaust particles (DEP) induces lung inflammation, the signaling pathways involved in the pathogenesis remain unclear. Toll-like receptors (TLRs) are generally accepted to be pathogen recognition receptors in mammalians. In the present study, we investigated the role of TLR-4 in DEP-induced lung inflammation and cytokine expression in the lung in TLR-4 point mutant (C3H/HeJ) mice and corresponding control (C3H/HeN) mice. Both the types of mice were randomized into four experimental groups that received vehicle or DEP (12 mg/kg body weight) by intratracheal instillation (n=8–10 in each group). Cellular profile of bronchoalveolar lavage (BAL) fluid, expressions of cytokines and chemokines in the lung, and circulatory fibrinogen levels were evaluated 24 h after the instillation.DEP challenge revealed a significant increase in the numbers of total cells and neutrophils in the BAL fluid as compared to vehicle challenge, however, the numbers were less in C3H/HeJ mice than in C3H/HeN mice. DEP exposure significantly induced the lung expression of interleukin (IL)-1β, keratinocyte chemoattractant (KC), and macrophage inflammatory protein (MIP)-1α when compared to vehicle challenge in both genotypes of mice. In the presence of DEP, the level of MIP-1α was significantly lower in C3H/HeJ mice than in C3H/HeN mice, however, the levels of IL-1β, KC, and fibrinogen showed opposite findings. These results suggest that TLR-4 is one of recognition receptors against DEP in the airways.

Keywords: Diesel exhaust particles; Toll-like receptor-4; Cytokine; Chemokine

Inhibition of endothelial nitric oxide synthase activity and suppression of endothelium-dependent vasorelaxation by 1,2-naphthoquinone, a component of diesel exhaust particles by Yang Sun; Keiko Taguchi; Daigo Sumi; Shigeru Yamano; Yoshito Kumagai (pp. 280-285).

Diesel exhaust particles contain redox-active quinones, such as 9,10-phenanthraquinone (9,10-PQ) and 1,2-naphthoquinone (1,2-NQ), which act as potent electron acceptors, thereby altering electron transfer on proteins. We have previously found that 9,10-PQ inhibits constitutive nitric oxide synthase (NOS) activity, by shunting electrons away from NADPH on the cytochrome P450 reductase domain of NOS, and thus suppresses acetylcholine (Ach)-induced vasorelaxation in the aortic ring. However, the effect of 1,2-NQ on endothelial NOS (eNOS) activity is still poorly understood. With the membrane fraction of cultured bovine aortic endothelial cells, we found that 1,2-NQ was a potent inhibitor of eNOS with an IC₅₀ value of 1.4 μM, whereas trans-1,2-dihydroxy-1,2-dihydronaphthalene (1,2-DDN), a redox-negative naphthalene analog of 1,2-NQ, did not show such an inhibitory action. Although 1,2-DDN (5 μM) did not affect Ach-mediated vasorelaxation, 1,2-NQ caused a significant suppression of Ach-induced endothelium-dependent vasorelaxation in the aortic ring. However, 1,2-NQ did not affect sodium nitroprusside-induced endothelium-independent vasorelaxation. These results suggest that 1,2-NQ is an environmental quinone that inhibits eNOS activity, thereby disrupting NO-dependent vascular tone.

Keywords: 1,2-Naphthoquinone; Nitric oxide; Endothelial nitric oxide synthase; Vasorelaxation; Diesel exhaust particles

Developmental and neurobehavioral effects of perinatal exposure to polychlorinated biphenyls in mice by Norio Sugawara; Kunihiko Nakai; Tomoyuki Nakamura; Takashi Ohba; Keita Suzuki; Satomi Kameo; Chieko Satoh; Hiroshi Satoh (pp. 286-292).

Because behavioral deficits associated with gestational exposure to polychlorinated biphenyls (PCBs) have been a concern, we studied the developmental and neurobehavioral effects of perinatal exposure to Aroclor 1254 (A1254), a commercial mixture of PCBs, in mice. The PCB mixture (A1254; 0, 6, 18, and 54 mg/kg body weight) was administered to pregnant mice (C57BL/6Cr) every 3 days by gavage from gestational day (GD) 6 to postnatal day (PND) 20. Compared with the control, treatment with A1254 did not alter the maternal body weight during the gestation and lactation periods. The body weight of the offspring did not differ among treatments. To assess the effects on offspring following such exposure, physical and neurobehavioral development (i.e., pinna detachment, hair growth, eye opening, incisor eruption, grasp reflex, righting reflex, walking, negative geotaxis, and cliff avoidance) was observed before weaning. At PND 7, poor adult-like responses in negative geotaxis were observed in all exposed groups. When the offspring were at 8-week old, the PCB-treated (18 mg/kg body weight) mice showed a decreased walking speed in the open-field test, and a prolonged time to reach the platform in the water maze test. Spontaneous locomotion activity was not affected by PCB exposure at 9 weeks . These results showed that perinatal exposure to PCBs produces several behavioral alterations in mice. Although dose-dependent changes were not observed, the neurobehavioral effects such as a decreased walking speed in the open-field test and a prolonged time to reach the platform in the water maze test remained in adulthood after the seeming recovery from the transient delay in development before weaning.

Keywords: PCBs; Neurobehavioral effect; Development; Aroclor1254; Perinatal exposure

Effects of di-(2-ethylhexyl)-phthalate and its metabolites on the lipid profiling in rat HRP-1 trophoblast cells by Yan Xu; Gregory T. Knipp; Thomas J. Cook (pp. 293-298).

The highly directional maternal-to-fetal transfer of essential fatty acids (EFAs) across the placenta plays a critical role in guiding proper fetal development. Exposure to xenobiotics that may alter the fetal supply of EFAs/lipids could lead to fetal toxicity. Since the placenta is the first fetal arising organ that regulates fetal fatty acid homeostasis, the fatty acid/lipid composition in the placenta may serve as an indicator of fetal composition. In this study, we investigated the effects of the peroxisome proliferator chemical di-(2-ethylhexyl)-phthalate (DEHP), a widely used plasticizer and ubiquitous environmental contaminant, and its selective metabolites, mono-(2-ethylhexyl)-phthalate (MEHP) and 2-ethylhexanoic acid (EHA) on the lipid metabolome in a rat HRP-1 trophoblast model. The concentrations of ten lipid classes (cholesterol esters, diacylglycerol, triacylglycerides, phosphatidylcholine, phosphatidylethanolamine, phosphatidylserine, lysophosphatidylcholine, cardiolipin, and sphingomyelin) were determined, as well as the individual fatty acid compositions, especially the ω-3 and ω-6 family of EFAs. The level of each lipid class was significantly increased upon exposure to the agents, with MEHP and EHA generally showing higher increases than DEHP. The same trends were observed in comparing the fatty acid compositions. For example, the ω-3/ω-6 fatty acids ratio did not change, although the levels of ω-3 and ω-6 fatty acids were significantly elevated upon exposure. These results suggest that DEHP and its metabolites can alter lipid metabolome in a rat placental cell line, implying that these compounds may contribute to aberrant placental EFA/lipid homeostasis caused by peroxisome proliferation, and potentially result in abnormal fetal development.

Keywords: Phthalates; Rat; Placenta; HRP-1; Lipomics; Metabolome

Absence of liver tumor-initiating activity of kojic acid in mice by Mitsuyoshi Moto; Taeko Mori; Miwa Okamura; Yoko Kashida; Kunitoshi Mitsumori (pp. 299-304).

In order to evaluate the tumor-initiating activity of kojic acid (KA) in mouse liver, an in vivo initiation assay in liver was performed using partially hepatectomized mice. Male ICR mice were fed on a basal diet (BD) containing 0 or 3% KA for 4 weeks, followed by distilled water (DW) containing 0 or 500 ppm phenobarbital (PB) for 13 weeks. Two weeks after the treatment with PB, two-thirds partial hepatectomy was preformed in all mice in order to enhance the regeneration and proliferating activities of the hepatocytes. In microscopic examinations, no proliferative lesion was observed in any of the groups. There were no differences in the number of γ-glutamyltransferase-positive cells, an expected marker for preneoplastic hepatocytes in mice, between the KA + DW and the KA + PB groups. In the immunohistochemical analyses of the proliferating activity of hepatocytes, significant increases in the labeling index of proliferating cell nuclear antigen (PCNA) were observed in the BD + PB and KA + PB groups as compared to the BD + DW group; however, no significant difference in the positivity of PCNA was observed between the BD + PB and the KA + PB groups. These results of the present study suggest the possibility that KA has no tumor-initiating activity in the liver of mice.

Keywords: Kojic acid; Initiation assay in the liver; Hepatocarcinogenicity; Genotoxicity; ICR mouse

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Archives of Toxicology (v.80, #5)