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Archives of Toxicology (v.85, #6)

Good news for sound science-based alternatives in liver by Bernward Garthoff (pp. 537-538).

Genetic variants in urinary bladder cancer: collective power of the “wimp SNPs” by Klaus Golka; Silvia Selinski; Marie-Louise Lehmann; Meinolf Blaszkewicz; Rosemarie Marchan; Katja Ickstadt; Holger Schwender; Hermann M. Bolt; Jan G. Hengstler (pp. 539-554).

In recent years, genome-wide association studies (GWAS) have identified more than 300 validated associations between genetic variants and risk of approximately 70 common diseases. A small number of rare variants with a frequency of usually less than 1% are associated with a strongly enhanced risk, such as genetic variants of TP53, RB1, BRCA1, and BRCA2. Only a very small number of SNPs (with a frequency of more that 1% of the rare allele) have effects of a factor of two or higher. Examples include APOE4 in Alzheimer’s disease, LOXL1 in exfoliative glaucoma, and CFH in age-related macular degeneration. However, the majority of all identified SNPs have odds ratios between 1.1 and 1.5. In the case of urinary bladder cancer, all known SNPs that have been validated in sufficiently large populations are associated with odds ratios smaller than 1.5. These SNPs are located next to the following genes: MYC, TP63, PSCA, the TERT-CLPTM1L locus, FGFR3, TACC3, NAT2, CBX6, APOBEC3A, CCNE1, and UGT1A. It is likely that these moderate risk or “wimp SNPs” interact, and because of their high number, collectively have a strong influence on whether an individual will develop cancer or not. It should be considered that variants identified so far explain only approximately 5–10% of the overall inherited risk. Possibly, the remaining variance is due to an even higher number of SNPs with odds ratios smaller than 1.1. Recent studies have provided the following information: (1) The functions of genes identified as relevant for bladder cancer focus on detoxification of carcinogens, control of the cell cycle and apoptosis, as well as maintenance of DNA integrity. (2) Many novel SNPs are far away from the protein coding regions, suggesting that these SNPs are located on distant-acting transcriptional enhancers. (3) The low odds ratio of each individual bladder cancer-associated SNP is too low to justify reasonable preventive measures. However, if the recently identified SNPs interact, they may collectively result in a substantial risk that is of preventive relevance. In addition to the “novel SNPs” identified by the recent GWAS, at least 163 further variants have been reported in relation to bladder cancer, although they have not been consistently validated in independent case–control series. Moreover, given that only 60 of these 163 “old SNPs” are covered by the SNP chips used in the recent GWAS, there are in principle 103 published variants still awaiting validation or disproval. In future, besides identifying novel disease-associated rare variants by deep sequencing, it will also be important to understand how the already identified variants interact.

Keywords: Genome-wide association study; SNP chip comparison; Bladder cancer risk; Genetic variant; Enzyme polymorphism; Validation; GSTM1

A physiologically based toxicokinetic modelling approach to predict relevant concentrations for in vitro testing by Hans Mielke; Lennart T. Anger; Markus Schug; Jan G. Hengstler; Ralf Stahlmann; Ursula Gundert-Remy (pp. 555-563).

Our study was performed in the context of an in vitro primary hepatic cell culture as an alternative for the in vivo cancerogenic bioassay. The 29 substances which are to be used in the in vitro primary hepatic cell culture have been tested in 2-year bioassays and a 14-day short term study. The aim of this modelling study was to simulate the concentration–time profile of the compounds when given by the oral route at the doses tested in the previous studies taking into account the percentage of the dose absorbed. The model contained seven tissue compartments with uptake from the gastrointestinal tract into the portal vein. Because the primary hepatic cell culture is metabolically competent and the primary interest was to model the concentration in the portal vein, the hepatic vein and the systemic circulation (blood) in the beginning we did not include elimination. Partitioning between blood and tissues was calculated according to a published biologically based algorithm. The substances’ kinetic profile differed according to their blood: tissue partitioning. Maximal concentrations in portal vein, hepatic vein and the blood depended mainly on the dose and the fraction absorbed which were the most critical parameters in this respect. Our study demonstrates an application of BPTK modelling for the purpose to simulate concentrations for planning the doses for an in vitro study. BPTK modelling seems to be a better approach than using data from in vitro studies on cytotoxicity.

Keywords: PBTK modelling; In vitro-in vivo extrapolation; Relevant concentrations in vitro

Inorganic arsenic causes cell apoptosis in mouse cerebrum through an oxidative stress-regulated signaling pathway by Cheng Chien Yen; Tsung Jung Ho; Chin Ching Wu; Chun Fang Chang; Chin Chuan Su; Ya Wen Chen; Tzyy Rong Jinn; Tien Hui Lu; Po Wen Cheng; Yi Chang Su; Shing Hwa Liu; Chun Fa Huang (pp. 565-575).

Arsenic pollution is a major public health problem worldwide. Inorganic arsenic (iAs) is usually more harmful than organic ones. iAs pollution increases the risk of human diseases such as peripheral vascular disease and cancer. However, the toxicological effects of iAs in the brain are mostly unclear. Here, we investigated the toxic effects and possible mechanisms of iAs in the cerebrum of mice after exposure to iAs (0.5 and 5 ppm (mg/l) As₂O₃, via the drinking water), which was the possible human exposed dose via the ingestion in iAs-contaminated areas, for 6 consecutive weeks. iAs dose-dependently caused an increase of LPO production in the plasma and cerebral cortex. iAs also decreased the reduced glutathione levels and the expressions of NQO1 and GPx mRNA in the cerebral cortex. These impairments in the cerebral cortex caused by iAs exposure were significantly correlated with the accumulation of As. Moreover, iAs induced the production of apoptotic cells and activation of caspase-3, up-regulation of Bax and Bak, and down-regulation of Mcl-1 in the cerebral cortex. Exposure to iAs also triggered the expression of ER stress-related genes, including GRP78, GRP94, and CHOP. Meanwhile, an increase of p38 activation and dephosphorylation of ERK1/2 were shown in the cerebral cortex as a result of iAs-exposed mice. These iAs-induced damages and apoptosis-related signals could be significantly reversed by NAC. Taken together, these results suggest that iAs-induced oxidative stress causes cellular apoptosis in the cerebrum, signaling of p38 and ERK1/2, and ER stress may be involved in iAs-induced cerebral toxicity.

Keywords: Inorganic arsenic; Apoptosis; Oxidative stress; ER stress; p38; ERK1/2

Analysis of arsenic metabolites in HepG2 and AS3MT-transfected cells by Takayuki Watanabe; Yuki Ohta; Ayano Mizumura; Yayoi Kobayashi; Seishiro Hirano (pp. 577-588).

It has been suggested that arsenic (+3 oxidation state) methyltransferase (AS3MT) plays a critical role in methylation of arsenic, and that arsenic–glutathione conjugate is a substrate for AS3MT-catalyzed methylation of arsenic. However, the mechanism of arsenic methylation in cells is not fully understood. Here, we have constructed T-REx-CHO-hAS3MTtr cells that transiently overexpress human AS3MT in response to tetracycline. The decreases in cell viability after exposure to sodium arsenite were greater in tetracycline-treated cells (tet(+) cells) than in untreated cells (tet(−) cells). Concentration of total cellular arsenic was significantly higher in tet(+) cells than in tet(−) cells. Speciation analyses of arsenic metabolites in whole cell lysates and cell culture medium were performed using both HepG2 cells and T-REx-CHO-hAS3MTtr cells. Speciation analyses of arsenic metabolites in lysates of T-REx-CHO-hAS3MTtr cells revealed that dimethylated arsenicals were the predominant arsenic metabolites in tet(+) cells, while methylated metabolites were not found in tet(−) cells. In contrast, less amount of methylated arsenic metabolites were found in the HepG2 cell lysates, and monomethylated trivalent arsenicals were the predominant methylated arsenic metabolites. Arsenate was found in the culture medium after 24 h culture with arsenite. A larger amount of arsenate was found in the culture medium of tet(+) or tet(−) cells compared to HepG2 cells. These findings indicated that AS3MT expression enhanced the cytotoxic effect of arsenite in tet(+) cells because these cells accumulated more arsenic metabolites than did the tet(−) cells, and accordingly, the tet(+) cells were more susceptible to arsenic than were the tet(−) cells. Oxidation–reduction of arsenic may be implicated in the toxic effects of arsenite.

Keywords: Arsenic; Glutathione; AS3MT; Metabolite speciation; ICP-MS

Reproductive toxicity parameters and biological monitoring in occupationally and environmentally boron-exposed persons in Bandırma, Turkey by Yalçın Duydu; Nurşen Başaran; Aylin Üstündağ; Sevtap Aydın; Ülkü Ündeğer; Osman Yavuz Ataman; Kaan Aydos; Yalçın Düker; Katja Ickstadt; Britta Schulze Waltrup; Klaus Golka; Hermann M. Bolt (pp. 589-600).

Boric acid and sodium borates have been considered as being “toxic to reproduction and development”, following results of animal studies with high doses. Experimentally, a NOAEL (no observed adverse effect level) of 17.5 mg B/kg-bw/day has been identified for the (male) reproductive effects of boron in a multigeneration study of rats, and a NOAEL for the developmental effects in rats was identified at 9.6 mg B/kg-bw/day. These values are being taken as the basis of current EU safety assessments. The present study was conducted to investigate the reproductive effects of boron exposure in workers employed in boric acid production plant in Bandırma, Turkey. In order to characterize the external and internal boron exposures, boron was determined in biological samples (blood, urine, semen), in workplace air, in food, and in water sources. Unfavorable effects of boron exposure on the reproductive toxicity indicators (concentration, motility, morphology of the sperm cells and blood levels of follicle-stimulating hormone (FSH), luteinizing hormone (LH), and total testosterone) were not observed. The mean calculated daily boron exposure (DBE) of the highly exposed group was 14.45 ± 6.57 (3.32–35.62) mg/day. These human exposures represent worst-case exposure conditions to boric acid/borates in Turkey. These exposure levels are considerably lower than exposures, which have previously led to reproductive effects in experimental animals. In conclusion, this means that dose levels of boron associated with developmental and reproductive toxic effects in animals are by far not reachable for humans under conditions of normal handling and use.

Keywords: Boron; Boric acid; Daily boron exposure; Reproductive toxicity; Semen; Biological monitoring

Excretion of 2,3-dihydroxy-propionamide (OH-PA), the hydrolysis product of glycidamide, in human urine after single oral dose of deuterium-labeled acrylamide by Eva C. Hartmann; Julia M. Latzin; Birgit K. Schindler; Holger M. Koch; Jürgen Angerer (pp. 601-606).

A dose of 0.99 mg d₃-acrylamide (d₃-AA) (13.2 μg/kg body weight) was ingested by a healthy male volunteer. Urine samples were collected over a period of 46 h after the intake and analyzed for the hydrolysis product of glycidamide (GA), 2,3-dihydroxy-propionamide (OH-PA), a metabolite of the toxicologically relevant oxidative AA metabolism pathway; 5.4% of the administered d₃-AA dose was eliminated as OH-PA within 46 h after ingestion. Therefore, OH-PA represents a major metabolite of the oxidative metabolism pathway. Elimination kinetics of OH-PA is similar to the oxidative metabolites N-acetyl-S-(2-carbamoyl-2-hydroxyethyl)-cysteine (GAMA) and N-acetyl-S-(1-carbamoyl-2-hydroxyethyl)-cysteine (iso-GAMA). The major excretion of d₃-OH-PA took place between 8 and 22 h with the highest urinary d₃-OH-PA concentration (c _max) of 69.3 μg/L urine, 18 h (t _max) postdose. OH-PA (5.4%), together with the other known urinary metabolites of the oxidative pathway GAMA (4.6%) and iso-GAMA (0.8%), represents 10.8% of the total AA dose. The share of the oxidative pathway metabolites is much smaller than the share of the reductive pathway metabolite N-acetyl-S-(2-carbamoylethyl)-cysteine (AAMA) that represents 51.7% of the ingested d₃-AA dose. However, this new quantitative human data on OH-PA together with the previous data on the other oxidative pathway metabolites are of special importance when evaluating the carcinogenic potential of AA and when comparing human data with data from animal studies.

Keywords: Acrylamide (AA); Glycidamide (GA); Biological monitoring; 2,3-Dihydroxy-propionamide (OH-PA); Metabolism; Toxicokinetics

Simple and quick method for whole-liver decellularization: a novel in vitro three-dimensional bioengineering tool? by Joery De Kock; Liesbeth Ceelen; Ward De Spiegelaere; Christophe Casteleyn; Paul Claes; Tamara Vanhaecke; Vera Rogiers (pp. 607-612).

Proof of principle of organ reengineering through the development of a transplantable recellularized liver graft was published recently. As the decellularization time of the rat liver took 72 h, loss of some key matrix proteins seemed inevitable. Here, we describe the development of a three-dimensional naturally derived liver scaffold with an intact microvascular system that is capable of withstanding fluid flows in the three hepatic circular systems and that is obtained within 60 min. For this purpose, whole rat livers were sequentially perfused with a selection of mild tensioactive substances to remove the cellular components while preserving the major extracellular matrix proteins, including laminin, collagen I, collagen IV, and fibronectin. In addition, we could show the presence of extracellular matrix–bound growth factor islets, important for cell engraftment, migration, proliferation, and differentiation. This easy to prepare scaffold could represent a remarkable tool in the bioengineering of complex three-dimensional in vitro systems for advanced preclinical drug development.

Keywords: Liver matrix; Organ decellularization; Biological liver scaffold; Three-dimensional scaffold

Perfluorooctane sulfonate increased hepatic expression of OAPT2 and MRP2 in rats by Wen-Guang Yu; Wei Liu; Li Liu; Yi-He Jin (pp. 613-621).

The toxicity of perfluorooctane sulfonate (PFOS), a persistent organic compound, is of great concern. Several studies have reported that PFOS decreases circulating thyroid hormone (TH) concentrations. However, the mechanisms involved remain to be determined. Female rats were exposed to (1) vehicle; (2) PFOS (0.2, 1.0, and 3.0 mg/kg); (3) propylthiouracil (PTU, 10 mg/kg); or (4) PTU (10 mg/kg) + PFOS (3.0 mg/kg) by gavage once a day for 5 consecutive days. Parameters including contents of total T4 (TT4) and total T3 (TT3) in both serum and bile, serum concentrations of transthyretin and thyroglobulin, as well as transcripts of transporters involved in hepatic uptake and efflux of T4 were determined in control and PFOS-exposed groups. TT4 and TT3 were also analyzed in PTU and PTU + PFOS groups in order to reflect the different hormone effects between PFOS, PTU, and PFOS + PTU. Results showed that serum TT4 and TT3 decreased, while bile TT4 and TT3 remained stable following PFOS exposure. Exposure to 3.0 mg/kg of PFOS significantly enhanced hepatic organic anion transporter OATP2 mRNA expression (1.43 times of control). Treatment with PFOS increased hepatic expression of multidrug resistance–associated protein MRP2, approximately 1.80 and 1.69 times of control in 1.0 and 3.0 mg/kg groups, respectively. Spearman’s correlation coefficients revealed that MRP2 mRNA expression correlated well with serum TT4 level (r = −0.528, P = 0.012). Serum thyroglobulin and transthyretin levels remained stable. Serum TT3, bile TT4, and bile TT3 were significantly different between PFOS and PTU groups. No significant differences of TT4 and TT3 in both serum and bile were observed between PTU and PTU + PFOS (P > 0.05). In conclusion, PFOS increased hepatic expression of OAPT2, which could possibly enhance hepatic uptake and metabolism of T4 in rats. PFOS-induced TT4 deficiency is mainly due to the extrathyroidal metabolism of T4, which is probably different from the classic goitrogen, PTU.

Keywords: Perfluorooctane sulfonate; Wistar rat; Thyroid hormone; Organic anion transporters 1 and 2; Multidrug resistance–associated protein 2; Propylthiouracil

Activation of group IVC phospholipase A₂ by polycyclic aromatic hydrocarbons induces apoptosis of human coronary artery endothelial cells by Patricia K. Tithof; Sean M. Richards; Mona A. Elgayyar; Fu-Minn Menn; Vijay M. Vulava; Larry McKay; John Sanseverino; Gary Sayler; Dawn E. Tucker; Christina C. Leslie; Kim P. Lu; Kenneth S. Ramos (pp. 623-634).

Exposure to environmental pollutants, such as polycyclic aromatic hydrocarbons (PAHs) found in coal tar mixtures and tobacco sources, is considered a significant risk factor for the development of heart disease in humans. The goal of this study was to determine the influence of PAHs present at a Superfund site on human coronary artery endothelial cell (HCAEC) phospholipase A₂ (PLA₂) activity and apoptosis. Extremely high levels of 12 out of 15 EPA high-priority PAHs were present in both the streambed and floodplain sediments at a site where an urban creek and its adjacent floodplain were extensively contaminated by PAHs and other coal tar compounds. Nine of the 12 compounds and a coal tar mixture (SRM 1597A) activated group IVC PLA₂ in HCAECs, and activation of this enzyme was associated with histone fragmentation and poly (ADP) ribose polymerase (PARP) cleavage. Genetic silencing of group IVC PLA₂ inhibited both ³H-fatty acid release and histone fragmentation by PAHs and SRM 1597A, indicating that individual PAHs and a coal tar mixture induce apoptosis of HCAECs via a mechanism that involves group IVC PLA₂. Western blot analysis of aortas isolated from feral mice (Peromyscus leucopus) inhabiting the Superfund site showed increased PARP and caspase-3 cleavage when compared to reference mice. These data suggest that PAHs induce apoptosis of HCAECs via activation of group IVC PLA₂.

Keywords: Atherosclerosis; Lipid signaling; Phospholipase A₂ ; Apoptosis; Anthracene; Benzo(a)pyrene; Acenapthalene; Benzo(a)anthracene; Benzo(b)fluoranthene; Benzo(k)fluoranthene; Benzo(g, h, i)perylene

Myosin heavy chain expression pattern as a marker for anabolic potency: desoxymethyltestosterone (madol), norandrostendione and testosterone repress MHC-IIb expression and stimulate MHC-IId/x expression in orchiectomized rat gastrocnemius muscle by S. Frese; M. Velders; B. Schleipen; W. Schänzer; W. Bloch; P. Diel (pp. 635-643).

Both 19-norandrostenedione (estr-4-ene-3,17-dione, NOR) and desoxymethyltestosterone (17alpha-methyl-5alpha-androst-2-en-17beta-ol, DMT or “madol”) are ‘designer steroids’ misused for doping purposes in the bodybuilding scene. We have previously characterized the pharmacological profile of madol and identified potential adverse side effects. The aim of this study was to investigate the anabolic potency of NOR, madol and the reference substance testosterone propionate (TP). Besides wet weight of the M.levator ani (LA), we examined the effects on muscle fiber type composition and myosin heavy chain (MHC) expression in the M.gastrocnemius (Gas) muscle as additional markers for anabolic potency. A Hershberger assay was performed, where orchiectomized (orchi) male Wistar rats were treated subcutaneously with NOR, madol, TP or vehicle control (all 1 mg/kg BW/day) for 12 days. Wet weights of the Gas, LA, prostate and seminal vesicle were examined to determine anabolic and androgenic effects. Fiber type composition of the Gas muscle was analyzed using ATPase staining, and MHC protein profiles were determined by silver stain and Western blot analysis. NOR and madol exhibited strong anabolic and weak androgenic potency by stimulating growth of the LA but not the prostate and seminal vesicle. Skeletal muscle fiber type composition characterized by ATPase staining was not significantly altered between the treatment groups, although there was a tendency toward lower levels of type IIB and increased type IIA fibers in all treatment groups relative to orchi. MHC protein expression determined by Western blot and silver stain analysis revealed that MHC IId/x was significantly up-regulated, while MHC IIb was significantly down-regulated in NOR, madol and TP groups relative to orchi. There were no significant differences for MHC IIa and MHC I expression between groups. Results suggest that the observed MHC expression shift could serve as a molecular marker to determine anabolic activity of anabolic steroids at least in skeletal muscle of orchi rats. The molecular mechanisms as well as the androgen-dependent regulation of MHC expression in intact skeletal muscle remain to be further investigated.

Keywords: Testosterone; Madol; Desoxymethyltestosterone; NORandrostendione; Skeletal muscle; Myosin heavy chain; Fiber type composition

Diphenyl diselenide induces apoptotic cell death and modulates ERK1/2 phosphorylation in human neuroblastoma SH-SY5Y cells by Thaís Posser; Mariane Trindade de Paula; Jeferson Luis Franco; Rodrigo B. Leal; João Batista T. da Rocha (pp. 645-651).

Diphenyl diselenide (PhSe)₂ is a synthetic organoselenium compound displaying glutathione peroxidase-like activity. Protective and antioxidant potential of (PhSe)₂ have been extensively investigated in in vivo and in vitro studies. In spite of this, there is a lack of studies addressed to the investigation of potential cytotoxic effect and signaling pathways modulated by this compound. Herein, we aimed to analyze the effects of 24-h treatment with (PhSe)₂ on cell viability and a possible modulation of signaling pathways in human neuroblastoma cell line SH-SY5Y. For this purpose, cells were incubated with (PhSe)₂ (0.3–30 μM) for 24 h and cell viability, apoptotic cell death and modulation of MAPKs (ERK1/2 and p38^MAPK), and PKC substrates phosphorylation was determined. (PhSe)₂ treatment significantly decreased cell viability and increased the number of apoptotic cells with induction of PARP cleavage. An increase in ERK1/2 phosphorylation was observed at (PhSe)₂ 3 μM. In contrast, higher concentrations of the chalcogenide inhibited ERK1/2, p38^MAPK and PKC substrate phosphorylation. Pre-treatment with ERK1/2 inhibitor, U0126, increased cell susceptibility to (PhSe)₂. Together, these data indicate a cytotoxic potential of (PhSe)₂ in a neuronal cell line, which appears to be mediated by the ERK1/2 pathway.

Keywords: SH-SY5Y cells; Diphenyl diselenide; MAPKs; ERK1/2; PKC

Global DNA methylation in the mouse liver is affected by methyl deficiency and arsenic in a sex-dependent manner by Keiko Nohara; Takashi Baba; Hikari Murai; Yayoi Kobayashi; Takehiro Suzuki; Yukiyo Tateishi; Michiyo Matsumoto; Noriko Nishimura; Tomoharu Sano (pp. 653-661).

Arsenic, a carcinogen, is assumed to induce global DNA hypomethylation by consuming the universal methyl donor S-adenosylmethionine (SAM) in the body. A previous study reported that a methyl-deficient diet (MDD) with arsenic intake greatly reduced global DNA methylation (the content of 5-methylcytosine) in the liver of male C57BL/6 mice. In the present study, we investigated the DNA methylation level, SAM content, and expression of DNA methyltransferases (DNMTs) in the liver of male and female C57BL/6 mice fed a methyl-sufficient diet (MSD), an MDD, or an MDD + arsenic. The DNA methylation level was accurately determined by measuring the content of genomic 5-methyldeoxycytidine (5medC) by high-performance liquid chromatography/electrospray ionization mass spectrometry (LC/ESI–MS) using stable-isotope-labeled 5medC and deoxycytidine (dC) as internal standards. The results of this study revealed that while the MDD and arsenic tended to reduce the genomic 5meC content in the male mice livers, the MDD + arsenic significantly increased the 5meC content in the female mice livers. Another unexpected finding was the small differences in 5meC content among the groups. The MDD and MDD + arsenic suppressed DNMT1 expression only in the male mice livers. In contrast, SAM content was reduced by the MDD and MDD + arsenic only in the livers of female mice, showing that the changes in 5meC content were not attributable to SAM content. The sex-dependent changes in 5meC content induced by methyl deficiency and arsenic may be involved in differences in male and female susceptibility to diseases via epigenetic modification of physiological functions.

Keywords: DNA methylation; Methyl deficiency; Arsenic; 5-Methylcytosine; S-adenosylmethionine; Sex dependence

Effects of amlodipine on TGF-β-induced Smad2, 4 expressions in adriamycin toxicity of rat mesangial cells by Yun-Jie Song; Jin Li; Xian-Fei Xie; Hui Wang; Qi-Xiong Li (pp. 663-668).

Transforming growth factor-β (TGF-β) is closely associated with progressive renal fibrosis. A central component of TGF-β-stimulated mesangial cell fibrogenesis is the TGF-β family-specific Smad signal transduction pathway. This study investigated the expression of TGF-β-receptor–activated Smad2, its common partner Smad4, and the phosphorylated Smad2 (p-Smad2) in adriamycin-induced toxicity of cultured rat mesangial cells. This in vitro study showed that amlodipine (10⁻⁹ to 10⁻⁵ mol/l) had no effect on the toxicity of rat mesangial cells induced by adriamycin in the absence of TGF-β1. However, amlodipine (10⁻⁷ to 10⁻⁵ mol/l) reduced the toxicity of rat mesangial cells induced by TGF-β1 in the absence of adriamycin; moreover, amlodipine (10⁻⁸ to 10⁻⁵ mol/l) significantly reduced adriamycin-induced cytotoxicity when it was given in combination with TGF-β1; amlodipine (10⁻⁶, 10⁻⁵ mol/l) had no effect on Smad2 mRNA and protein expression induced by adriamycin + TGF-β1, but it (10⁻⁶, 10⁻⁵ mol/l) dramatically inhibited the down-regulation of p-Smad2 protein expression as well as Smad4 mRNA and protein expression induced by adriamycin + TGF-β1 in rat mesangial cells. Present study shows that amlodipine exerts a significant inhibition on adriamycin-induced toxicity in rat mesangial cells by affecting the expression of TGF-β/Smad signaling intermediates p-Smad2 and Smad4.

Keywords: Amlodipine; Adriamycin; Transforming growth factor-β; Smad; Mesangial cells

PARP-1 expression and activity in primary human lung cells by Mohamed Ahmad; Abdelrahman Torky; Felix Glahn; Robert J. Scheubel; Heidi Foth (pp. 669-679).

Activation of poly(ADP-ribose) polymerase-1 (PARP-1) in response to DNA damage is an important mechanism to keep homeostasis or to trigger apoptosis. The expression and function of (PARP-1) was studied in primary cells cultured from human lung. Normal human bronchial epithelial cells (NHBEC) and peripheral lung cells (PLC) from lung cancer patients were grown as explant cultures and were followed over a period of 12 weeks. PARP-1 protein was expressed in all explant cultures from bronchial epithelium. The levels of PARP protein differed between individuals by a factor of 2.3 in the first explant. Three cases were followed for more than 100 days. The expression levels varied intra-individually by a factor of 1.3–1.4 over this time period. PARP-1 activity was determined immunohistochemically after induction of DNA damage with H₂O₂ (0.05–0.3 mM, 5 min). The fluorescence signal for ADP-ribose polymers attached to chromatin proteins correlated well with the concentration of H₂O₂. PARP-1 activity differed by a factor of 3.1 in NHBECs obtained from the first generation of explants from 11 cases. PARP-1 activity is present in NHBECs until the 8th and in PLCs until the 12th week and declined to about half of the start level. Primary cultures of NHBECs and PLC are suitable to study the effect of external factors on PARP-1 expression and function.

Keywords: NHBEC; PLC; Long-term cultivation; PARP-1 protein expression; PARP-1 activity; Inter-individual variation

Production of liver preneoplasia and gallbladder agenesis in turkey fetuses administered diethylnitrosamine by Gary M. Williams; Klaus D. Brunnemann; Michael J. Iatropoulos; Daniel J. Smart; Harald G. Enzmann (pp. 681-687).

The in ovo carcinogenicity assessing (IOCA) assay was used to examine the morphological changes in fetal turkey livers caused by the DNA-reactive carcinogen diethylnitrosamine (DEN). Fertilized turkey eggs were allocated into 3 groups: nondosed control (NDC), vehicle (water) control (VC) and DEN-dosed. At day 0, the fertilized eggs of the dosed groups were injected with 1 (LD) or 4 (HD) mg/egg (about 12.5 or 50 mg/kg egg) of DEN and the VC were injected with water. All eggs were allowed to incubate at 37°C and 60% humidity for 24 days. The fetal livers were collected and processed for histopathological evaluation (H&E staining). Typical survival rates were 82% for the NDC, 50% for the VC and 16–65% for the DEN-dosed fetuses. No difference in histology was found between NDC and VC control groups. Both DEN concentrations produced dose-related liver findings consisting of foci of altered hepatocytes (FAH), which had assumed a tubular cord (glandular) pattern, and in HD DEN group the FAH assumed a tumor-like morphology. In addition, the high DEN dose produced gallbladder agenesis. Thus, DEN produced both hepatocellular transformation (FAH) similar to preneoplastic microscopic changes in adult rodents, reflecting disruption of the fetal processes of differentiation and proliferation, and also teratogenicity (gallbladder agenesis).

Keywords: In ovo carcinogenicity assessing assay in turkey; Diethylnitrosamine; Fetal liver differentiation; Proliferation and preneoplasia; Gallbladder agenesis

In vitro aneugenic effects of the fungicide thiabendazole evaluated in human lymphocytes by the micronucleus assay by Alfredo Santovito; Piero Cervella; Massimiliano Delpero (pp. 689-693).

Thiabendazole is a benzimidazole-derived compound widely employed in agriculture as anthelmintic and fungicide. It is also used as a post-harvest fungicide for imported citrus fruits during transport and storage, and thus, it was found at high concentration in fruits and vegetables. Several studies have analyzed the potential genotoxic effect of thiabendazole on different prokaryotic and eukaryotic systems, but in many cases, results were contradictory. In the present study, the genotoxic potential of thiabendazole have been evaluated, by micronucleus assay in freshly isolated human peripheral lymphocytes. The cells were incubated with 0.5, 5 and 50 μg/ml concentrations of the tested substance for 48 h at 37°C. Mitomycin C at final concentration of 0.01 μg/ml culture was used as a positive control. The results indicated that the thiabendazole significantly (P < 0.05) increased the micronucleus frequency compared with the negative control in all treatment concentrations, indicating a potential aneugenic hazard of thiabendazole in cultured human peripheral lymphocytes. The cytokinesis-block proliferation index value, however, was not decreased significantly compared with the negative control. Significant (P < 0.05) differences in the micronuclei frequency were also found between the lower dose (0.5 μg/ml) and the other two analyzed doses of thiabendazole. In contrast, no differences were found between 5 and 50 μg/ml of thiabendazole and between DMSO and negative control. Finally, control cultures treated with the known mutagen MMC showed a very consistent increase in MN with respect to the negative controls.

Keywords: Pesticide; Genotoxicity; Cytogenetic study; Benzimidazole

Cytotoxicity of zinc oxide (ZnO) nanoparticles is influenced by cell density and culture format by Boon Chin Heng; Xinxin Zhao; Sijing Xiong; Kee Woei Ng; Freddy Yin-Chiang Boey; Joachim Say-Chye Loo (pp. 695-704).

A parameter that has often been overlooked in cytotoxicity assays is the density and confluency of mammalian cell monolayers utilized for toxicology screening. Hence, this study investigated how different cell seeding densities influenced their response to cytotoxic challenge with ZnO nanoparticles. Utilizing the same volume (1 ml per well) and concentration range (5–40 μg/ml) of ZnO nanoparticles, contradictory results were observed with higher-density cell monolayers (BEAS-2B cells) obtained either by increasing the number of seeded cells per well (50,000 vs. 200,000 cells per well of 12-well plate) or by seeding the same numbers of cells (50,000) within a smaller surface area (12-well vs. 48-well plate, 4.8 vs. 1.2 cm², respectively). Further experiments demonstrated that the data may be skewed by inconsistency in the mass/number of nanoparticles per unit area of culture surface, as well as by inconsistent nanoparticle to cell ratio. To keep these parameters constant, the same number of cells (50,000 per well) were seeded on 12-well plates, but with the cells being seeded at the edge of the well for the experimental group (by tilting the plate) to form a dense confluent monolayer, as opposed to a sparse monolayer for the control group seeded in the conventional manner. Utilizing such an experimental set-up for the comparative evaluation of four different cell lines (BEAS-2B, L-929, CRL-2922 and C2C12), it was observed that the high cell density monolayer was consistently more resistant to the cytotoxic effects of ZnO nanoparticles compared to the sparse monolayer for all four different cell types, with the greatest differences being observed above a ZnO concentration of 10 μg/ml. Hence, the results of this study demonstrate the need for the standardization of cell culture protocols utilized for toxicology screening of nanoparticles, with respect to cell density and mass/number of nanoparticles per unit area of culture surface.

Keywords: Cell density; Confluent; Nanoparticle; Toxicology; Viability; Zinc Oxide

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Archives of Toxicology (v.85, #6)