Effect of multiple doses of styrene and R-styrene oxide on CC10, bax, and bcl-2 expression in isolated Clara cells of CD-1 mice

Harvilchuck, Jill A.; Carlson, Gary P.

doi:10.1016/j.tox.2009.02.016

Cited by 9 publications

(4 citation statements)

References 25 publications

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“…The RT‐PCR reaction conditions were: 1 cycle of 5 min at 94°C for enzyme activation, 25 cycles at 94°C for 30 s, 58°C for Bcl‐2 , 56°C for Bax or 60°C for β‐ actin for 30 s, then 72°C for 30 s. The PCR products were electrophoresed on 1% agarose gels in TAE buffer and visualized by staining with ethidium bromide (0.5 μg/mL). A gel electrophoresis image analysis system (Jetta Technology, JD801, China) was used to test the absorbance value of every band 17. The Bcl‐2 ‐, Bax ‐specific gene primers and the internal control gene primers (β‐ actin ) were: Bcl‐2 sense, 5′‐CTACCGTCGTGACTTCGC‐3′, and Bcl‐2 antisense, 5′‐CCTATTGCCTCCGACCCT‐3′; Bax sense, 5′‐AAAGTAGAAGAGGG CAACCAC‐3′, and Bax antisense, 5′‐AACCACCTGCGTAGGACC‐3′; β‐ actin sense, 5′‐TTCCAGCCCTCCTTCCTG‐3′, and β‐ actin antisense, 5′‐GCCCGACTCGTCA TACTCC‐3′.…”

Section: Methodsmentioning

confidence: 99%

“…A gel electrophoresis image analysis system (Jetta Technology, JD801, China) was used to test the absorbance value of every band. 17 The Bcl-2-, Bax-specific gene primers and the internal control gene primers (b-actin) were: Bcl-2 sense, 5 0 -CTACCGTCGTGACTT CGC-3 0 , and Bcl-2 antisense, 5 0 -CCTATTGCCTCCGACCCT-3 0 ; Bax sense, 5 0 -AAAGTAGAAGAGGG CAACCAC-3 0 , and Bax antisense, 5 0 -AACCACCTGCGTAGGACC-3 0 ; b-actin sense, 5 0 -TTCC AGCCCTCCTTCCTG-3 0 , and b-actin antisense, 5 0 -GCCCGACTC GTCA TACTCC-3 0 .…”

Section: Fcm Analysismentioning

confidence: 99%

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Comparison of MTT assay, flow cytometry, and RT‐PCR in the evaluation of cytotoxicity of five prosthodontic materials

et al. 2010

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In the present study, the cytotoxic effects of five prosthodontic materials on the L929 cell line were assessed by flow cytometry (FCM), reverse transcription PCR (RT-PCR), and MTT (3-(4,5-dimethylthiazole-2-yl)-2,5-diphenyltetrazoli-umbromide) assay. The cells were treated with eluates resin (RE), pressable ceramics (PC), Co-Cr alloy-porcelain (CC), Ni-Cr alloy-porcelain (NC), and diatomite ceramics (DC). The cytotoxicity of all the materials tested by the MTT assay was grade 1. By FCM analysis, apoptosis rates of DC and PC were low, with no significant difference from the control (p > 0.05). The rest of the groups induced much higher apoptosis rates (p < 0.05), with the highest in the RE group. The necrotic cell levels of RE was also significantly increased (p < 0.05). Bcl-2 and Bax mRNA expression were determined by RT-PCR, and the Bax/Bcl-2 ratio in the DC and PC groups were not significantly different from the control (p > 0.05), whereas CC, NC, and RE groups showed significant differences (p < 0.05). Taken together, the results suggest that FCM and RT-PCR analyses can supplement the traditional MTT assay in evaluating the cytotoxicity of prosthodontic materials for selecting highly biocompatible materials.

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Section: Methodsmentioning

confidence: 99%

Section: Fcm Analysismentioning

confidence: 99%

Comparison of MTT assay, flow cytometry, and RT‐PCR in the evaluation of cytotoxicity of five prosthodontic materials

et al. 2010

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“…The cytotoxicity from styrene and SO exposure is thought to be similar to oxidative stress-induced conditions caused by oxidizing protein thiols [4]. Primary cerebellar granule neurons and human neuroblastoma cells exposed to SO (0.3 to 1 mM) induced apoptosis, which can be triggered by oxidative stress [1, 10–12]. There is also evidence that exposure of primary striatal neurons to SO induces synaptic impairments [2], which might be the reflection of morphological alteration of the neuronal cytoskeleton.…”

Section: Introductionmentioning

confidence: 99%

Assessment of Styrene Oxide Neurotoxicity Using In Vitro Auditory Cortex Networks

Gopal

Moore

et al. 2011

ISRN Otolaryngology

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Styrene oxide (SO) (C 8 H 8 O), the major metabolite of styrene (C 6 H 5 CH=CH 2 ), is widely used in industrial applications. Styrene and SO are neurotoxic and cause damaging effects on the auditory system. However, little is known about their concentrationdependent electrophysiological and morphological effects. We used spontaneously active auditory cortex networks (ACNs) growing on microelectrode arrays (MEA) to characterize neurotoxic effects of SO. Acute application of 0.1 to 3.0 mM SO showed concentration-dependent inhibition of spike activity with no noticeable morphological changes. The spike rate IC 50 (concentration inducing 50% inhibition) was 511 ± 60 µM (n = 10). Subchronic (5 hr) single applications of 0.5 mM SO also showed 50% activity reduction with no overt changes in morphology. The results imply that electrophysiological toxicity precedes cytotoxicity. Fivehour exposures to 2 mM SO revealed neuronal death, irreversible activity loss, and pronounced glial swelling. Paradoxical "protection" by 40 µM bicuculline suggests binding of SO to GABA receptors.

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“…The two cell types may be differentially susceptible to various carcinogens. Murine Clara cells have been suggested to be specifically sensitive to the toxic or carcinogenic effects of coumarin, naphthalenes, N-nitrosobis-chloroethylureas, styrene, and trichloroethylene [Boyd and Reznik-Schüller, 1984;Rehm et al, 1991a;Born et al, 1998;Green, 2000;Cruzan et al, 2002;Harvilchuck and Carlson, 2009;Lin et al, 2009]. Mouse Type II cells have been described to be the target for tumor formation by N-nitrosoethylurea and 4-(methylnitrosamino)21-(3-pyridyl)21-butanone [Rehm et al, 1991b;Belinsky et al, 1992].…”

Section: Introductionmentioning

confidence: 99%

Micronucleus assay for mouse alveolar Type II and Clara cells

Lindberg

Falck

Catalán

et al. 2009

Environ and Mol Mutagen

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The objective of our study was to develop a micronucleus (MN) assay for detecting genotoxic damage after inhalation exposure in mouse alveolar Type II and Clara cells, potential target cells for lung carcinogens. Ten male C57BL/6J mice were exposed to ethylene oxide (630 mg/m(3)) for 4 hr via inhalation; 10 unexposed mice serving as controls. 72 hr after the exposure, Clara cells and alveolar Type II cells were isolated using two different methods. Method 1 included a 15-min trypsin lavage and a 2-hr incubation of cell suspension. Method 2 involved a 30-min trypsin lavage, Percoll gradient centrifugation, and a 48-hr incubation for cell attachment. Nitro blue tetrazolium (NBT) -staining was applied to distinguish Clara cells. The frequency of micronuclei (MNi) was scored in NBT-negative cells (defined as Type II cells in Method 2) and NBT-positive cells (Clara cells). To detect possible differences between the techniques, MNi in Clara cells were analyzed from samples prepared by both methods. With Method 2, a clear increase in the mean frequency of micronucleated cells was seen in the exposed mice as compared with the controls, for both alveolar Type II and Clara cells. However, no significant increase in MN frequency was seen in Clara cells analyzed from samples prepared by Method 1. Based on our findings, mouse alveolar Type II and Clara cells seem to be suitable for MN analysis in studies aimed at identifying genotoxic lung carcinogens. Both alveolar Type II and Clara cells can be isolated using Method 2.

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Effect of multiple doses of styrene and R-styrene oxide on CC10, bax, and bcl-2 expression in isolated Clara cells of CD-1 mice

Cited by 9 publications

References 25 publications

Comparison of MTT assay, flow cytometry, and RT‐PCR in the evaluation of cytotoxicity of five prosthodontic materials

Comparison of MTT assay, flow cytometry, and RT‐PCR in the evaluation of cytotoxicity of five prosthodontic materials

Assessment of Styrene Oxide Neurotoxicity Using In Vitro Auditory Cortex Networks

Micronucleus assay for mouse alveolar Type II and Clara cells

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