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Archives of Toxicology (v.83, #2)
A plea for rigorous and honest science: false positive findings and biased presentations in epidemiological studies
by Peter Morfeld (pp. 105-106).
Arsenic-induced suicidal erythrocyte death by Hasan Mahmud; Michael Föller; Florian Lang (pp. 107-113).
Environmental exposure to arsenic has been associated with anemia, which could result from suicidal erythrocyte death or eryptosis, characterized by cell shrinkage and phosphatidylserine exposure at the erythrocyte surface. Eryptosis is triggered by increase in cytosolic Ca2+ concentration, ceramide and energy depletion. The present experiments explored, whether arsenic stimulates eryptosis. According to annexin V-binding, arsenic trioxide (7 μM) within 48 h significantly increased phosphatidylserine exposure of human erythrocytes without inducing hemolysis. According to forward scatter, arsenic trioxide (7 μM) significantly decreased cell volume. Moreover, Fluo3-fluorescence showed that arsenic (10 μM) significantly increased cytosolic Ca2+ concentration. According to binding of respective fluorescent antibodies, arsenic trioxide (10 μM) significantly increased ceramide formation. Arsenic (10 μM) further lowered the intracellular ATP concentration. Removal of extracellular Ca2+ or inhibition of the Ca2+-permeable cation channels with amiloride blunted the effects of arsenic on annexin V-binding and cell shrinkage. In conclusion, arsenic triggers suicidal erythrocyte death by increasing cytosolic Ca2+ concentration, by stimulating the formation of ceramide and by decreasing ATP availability.
Keywords: Cell volume; Annexin; Eryptosis; Calcium; Phosphatidylserine
Modulation of the GSTT1 activity by the GSTM1 phenotype in a sample of Italian farm-workers by Maria Fuciarelli; Annamaria Caccuri; Maria De Francesca; Flavia Ferazzoli; Sara Piacentini; Flavia Porreca (pp. 115-120).
Glutathione S-transferase (GST) isozymes catalyze nucleophilic attack by reduced Glutathione (GSH) on a variety of electrophilic compounds and play a central role in biotransformation of xenobiotics (Hayes et al., Annu Rev Pharmacol Toxicol 45:51–88, 2005). We performed a case–control study to evaluate the GSTM1 and GSTT1 polymorphisms and to investigate if exposure to pesticides conditions the GSTT1 activity level in 115 healthy controls and 90 farm-workers exposed to pesticides. Polymorphisms were investigated using a GSTM1 or a GSTT1-specific PCR. Enzyme activity was measured by means of DCM as co-substrate, as described by Bruhn et al. (Biochem Pharmacol 56:1189–1193, 1998). There was no significant difference between the farm-workers and the healthy controls regarding the distribution of various alleles of the GSTM1 and GSTT1 genes and the GSTT1 enzyme activity. In farm-workers, the GSTM1 null genotype was associated with a significant increase of GSTT1 activity, suggesting a regulative mechanism common to GSTM1 and GSTT1 enzymes after exposure to xenobiotics.
Keywords: Glutathione S-transferase M1; Glutathione S-transferase T1; Genetic polymorphism; Xenobiotics; Farm-workers
Active extracts of wild fruiting bodies of Antrodia camphorata (EEAC) induce leukemia HL 60 cells apoptosis partially through histone hypoacetylation and synergistically promote anticancer effect of trichostatin A by Mei-Chin Lu; Ying-Chi Du; Jiunn-Jye Chuu; Shiuh-Lin Hwang; Pao-Chuan Hsieh; Chih-Sheng Hung; Fang-Rong Chang; Yang-Chang Wu (pp. 121-129).
The endemic species of Antrodia camphorate (AC) is a promising chemotherapeutic drug for cancer. We found that the ethanol extract from wild fruiting bodies of Antrodia camphorata (EEAC) could induce HL 60 cells apoptosis via histone hypoacetylation, up-regulation of histone deacetyltransferase 1 (HDAC 1), and down-regulation of histone acetyltransferase activities including GCN 5, CBP and PCAF in dose-dependent manner. In combination with histone deacetylase inhibitor, trichostatin A (TSA), did not block EEAC-induced apoptosis. Interestingly, combined treatment (100 nM of TSA and 100 μg/ml EEAC) caused synergistic inhibition of cell growth and increase of apoptotic induction. EEAC could effectively increase the cytotoxic sensitivity of TSA through the up-regulation of DR5 and NFκB activation. In this present study, bioassay-guided fractionation of EEAC led to a major active compound, zhankuic acid A, as the bioactive marker. Moreover, our findings may represent an experimental basis for developing EEAC as a potential chemotherapeutic adjuvant.
Keywords: Antrodia camphorata (AC) ; Death receptor (DR); Histone acetylation; Nuclear factor κB (NFκB); Trichostatin A (TSA); Zhankuic acid A
Distinct subtypes of urinary bladder epithelial cells with inducible and non-inducible cytochrome P450 1A1 by Sabine Plöttner; Silvia Selinski; Hermann M. Bolt; Gisela H. Degen; Jan G. Hengstler; Peter H. Roos; Wolfram Föllmann (pp. 131-138).
Cultured primary porcine urinary bladder epithelial cells (PUBEC) represent an adequate and easy to handle in vitro system for studies of urothelial toxicity. PUBEC maintain in vivo-like metabolic activities and physiological functions. They express inducible cytochrome P4501A isoenzymes, which are of particular relevance, since they contribute to activation of bladder carcinogens. A possible drawback of PUBEC is their isolation from common domestic pigs that do not represent an inbred strain. In order to further establish PUBEC as a standard in vitro toxicity test system we analysed possible interindividual differences in CYP1A1 inducibility. Interestingly, we observed by flow cytometry that PUBEC obtained from individual pigs consist of two distinct subpopulations with inducible and non-inducible cells. A strong, concentration-dependent CYP1A1 induction was observed in the responsive subpopulation when incubated with benzo[a]pyrene (B[a]P) in a concentration range between 1 and 10 μM. In contrast, no CYP1A1 induction was obtained in the non-responsive subpopulation up to the highest tested concentrations of 100 μM. The fraction of responsive cells showed large interindividual differences ranging from 10 to 65% of the total cell number. For practical purposes it might be reasonable to analyse pools of PUBEC from five pigs which substantially reduce batch to batch variability. In conclusion, we have identified two functionally distinct subpopulations of urinary bladder epithelial cells. It will be interesting to study whether the CYP1A inducible subtype is more susceptible to bladder carcinogens.
Keywords: PUBEC; CYP1A1; Benzo[a]pyrene; Subpopulations; Inducibility
Carnitine deficiency: a possible risk factor in paracetamol hepatotoxicity by Hossam M. M. Arafa (pp. 139-150).
We have addressed in the current study the postulate whether or not carnitine deficiency would represent a risk factor in hepatotoxicity. Carnitine-deficient male Swiss albino rats were obtained following administration of d-carnitine (500 mg/kg, IP) for 10 consecutive days. Serum and liver carnitine levels, both total and free, were assessed to confirm carnitine depletion. Hepatotoxicity was induced by challenging animals with a single dose of paracetamol (1 g/kg, IP). Serum tumor necrosis factor (TNF-α) concentration, and serum activities of aspartate amino transferase (AST), alanine amino transferase (ALT) and alkaline phosphatase (ALP) were undertaken as biomarkers for toxicity. Liver contents of reduced glutathione (GSH), malondialdehyde (MDA), total nitric oxide (NO) and myeloperoxidase (MPO) activities were also investigated. Histopathological examination of liver sections was achieved to confirm the biochemical alterations. d-carnitine altered all biochemical markers and also induced mild tissue inflammation with dilatation and congestion of central and portal veins. Paracetamol produced an obvious hepatotoxicity model that was well characterized biochemically and morphologically. Combined administration of d-carnitine and paracetamol synergistically provoked marked toxicity that was more profound than either agent given alone. The present work was further extended to elucidate any hepatoprotective effect of carnitine supplementation in such toxicity paradigm. It was apparent that l-carnitine notably ameliorated all biochemical markers and also mitigated the gross histologic alterations induced by paracetamol. Data obtained so far would suggest that carnitine deficiency could possibly be a sequela as well as a causative clue for paracetamol hepatotoxicity.
Keywords: Carnitine deficiency; d-carnitine; l-carnitine; Paracetamol; Hepatotoxicity; TNF-α; NO; AST; ALT; ALP
ER-stress caused by accumulated intracistanal granules activates autophagy through a different signal pathway from unfolded protein response in exocrine pancreas cells of rats exposed to fluoride by Mie Ito; Hiroshi Nakagawa; Toshiya Okada; Syuichi Miyazaki; Saburo Matsuo (pp. 151-159).
In rat exocrine pancreas cells, fluoride treatment causes autophagy resulting from intracisternal granule accumulation. Excessive autophagy might promote a type of programmed cell death different from apoptosis. To clarify how fluoride-induced autophagy and subsequent cell death occurs, we investigated morphological and biochemical changes in exocrine pancreas cells of rats subcutaneously injected with NaF saline solution at 20 mg/kg dose twice daily for 4 days. Intracisternal granule, excessive autophagy and ribosomal degranulation were observed in fluoride-exposed cells, occasionally with necrotic changes. Fluoride-induced rER-stress increased eIF-2α phosphorylation and CHOP expression, but did not affect GRP78. Spliced XBP-1 expression was decreased in damaged cells. These findings indicate that rER-stress by intracisternal granule accumulation lead to autophagy in exocrine pancreas cells without UPR, suggesting that signal process of autophagy differs from that of UPR-apoptosis. It is likely that intense degranulation is a turning point that damaged cells change over from autophagy, cell-protective process, to cell-death process.
Keywords: Autophagy; Cell death; Fluoride; Exocrine pancreas cell; UPR; XBP-1
Mechanisms of estrogen-induced effects in avian reproduction caused by transovarian application of a xenoestrogen, diethylstilbestrol by Ryo Kamata; Fujio Shiraishi; Tokukazu Izumi; Shinji Takahashi; Akira Shimizu; Hiroaki Shiraishi (pp. 161-171).
To clarify breeding failure in avian species caused by the estrogenicity of chemicals, alterations in the reproductive systems of Japanese quail exposed in ovo to a xenoestrogen were investigated. An injection of diethylstilbestrol (DES) into the yolk before incubation decreased, after sexual maturation, egg-laying performance of female quails, which accompanied inducing abnormal development of the oviducts. All females treated with 50 ng DES/g of egg did not lay eggs, while 0.5–5 ng DES/g reduced egg weight and eggshell strength and thickness. In the uterus (shell gland), the mRNAs for calcium regulating factors, osteopontin and calbindin D28 K, were reduced dose-dependently by DES. Scanning electron microscopy showed that shell thinning was pronounced in the mammillary and cuticular layers of the eggshell, regions where osteopontin proteins are reportedly located. These indicate that transovarian exposure to xenoestrogens causes malformation and dysfunction of the oviducts, where calcium regulating molecules could play key roles in eggshell thinning.
Keywords: Avian reproductive disorder; Diethylstilbestrol; Eggshell thinning; Osteopontin; Calbindin D28K; Japanese quail
Hepatocarcinogenic susceptibility of rasH2 mice to troglitazone in a two-stage hepatocarcinogenesis model by Meilan Jin; Yukie Saekusa; Yasuaki Dewa; Jihei Nishimura; Sayaka Matsumoto; Makoto Shibutani; Keiji Hasumi; Kunitoshi Mitsumori (pp. 173-181).
Six-week-old rasH2 mice were injected intraperitoneally with N-diethylnitrosamine (DEN) after partial hepatectomy and administrated 0 or 6,000 ppm troglitazone (TRG) for 10 weeks. Relative liver weight of females increased significantly in the DEN + TRG group compared to the DEN-alone group. The numbers of γ-glutamyltranspeptidase- and proliferating cell nuclear antigen (PCNA)-positive cells tended to increase in both the sexes in the DEN + TRG group; however, these changes were not significantly different from those in the DEN-alone group. Levels of gene expressions for vascular endothelial growth factor (VEGF) and VEGFB (related to angiogenesis), tropomyosin 1 (Tpm1) and transforming growth factor-β (related to ras/MAPK cascade activation), and PCNA (related to cell proliferation) in females were significantly higher in the DEN + TRG than in the untreated control group but not in the DEN-alone group. Only Tpm1 gene had significantly higher expression in the DEN + TRG group than in the DEN-alone group. These results suggest that rasH2 mice are not susceptible to TRG in a two-stage hepatocarcinogenesis model.
Keywords: Troglitazone; PPAR agonist; rasH2 mouse
Threshold dose of piperonyl butoxide that induces reactive oxygen species-mediated hepatocarcinogenesis in rats by Masako Muguruma; Masaomi Kawai; Yasuaki Dewa; Jihei Nishimura; Yukie Saegusa; Hironobu Yasuno; Meilan Jin; Sayaka Matsumoto; Masayoshi Takabatake; Katsuhiko Arai; Kunitoshi Mitsumori (pp. 183-193).
To determine the threshold dose of piperonyl butoxide (PBO) that induces hepatocellular tumor-promoting effects, reactive oxygen species (ROS) generation, and drug-metabolizing enzymes that protect against ROS generation, partial hepatectomized rats were fed diets containing 0, 0.015, 0.03, 0.06, 0.125, 0.25, or 0.5% PBO after an i.p. injection of N-diethylnitrosamine (DEN) to initiate hepatocarcinogenesis. Histopathologically, Glutathione S-transferase placental form (GST-P)-positive foci were significantly increased in a dose-dependent manner in rats given 0.25% PBO or higher. The formation of microsomal ROS in the liver was significantly increased in 0.25 and 0.5% PBO. Real-time RT-PCR showed that the expression of the CYP1A1, UDPGTr-2, and Mrp3 genes was significantly upregulated in rats given 0.03% PBO or higher. These results suggest that 0.25% is the threshold dose of PBO that induces ROS-mediated hepatocarcinogenesis in rats, although the CYP1A1 gene that is related to ROS generation and the UDPGTr-2 and Mrp3 genes that are involved in protection against ROS were induced in the livers of rats even at a PBO dose of 0.03%.
Keywords: Piperonyl butoxide; Nongenotoxic carcinogen; Threshold; CYP1A1; UDPGTr-2; Mrp3