Somatic alterations in mitochondrial DNA produce changes in cell growth and metabolism supporting a tumorigenic phenotype.

There have been many reports of mitochondrial DNA (mtDNA) mutations associated with human malignancies. We have observed allelic instability in UV-induced cutaneous tumors at the mt-Tr locus encoding the mitochondrial tRNA for arginine. We examined the effects of somatic alterations at this locus by modeling the change in a uniform nuclear background by generating cybrids harboring allelic variation at mt-Tr. We utilized the naturally occurring mtDNA variation at mt-Tr within the BALB/cJ (BALB) and C57BL/6J (B6) strains of Mus musculus to transfer their mitochondria into a mouse ρ(0) cell line that lacked its own mtDNA. The BALB haplotype containing the mt-Tr 9821insA allele produced significant changes in cellular respiration (resulting in lowered ATP production), but increased rates of cellular proliferation in cybrid cells. Furthermore, the mtDNA genotype associated with UV-induced tumors endowed the cybrid cells with a phenotype of resistance to UV-induced apoptosis and enhanced migration and invasion capabilities. These studies support a role for mtDNA changes in cancer.

Application habits and practices of regular sunscreen users in the United States: Results of an online survey.

A nationwide online survey assessed claimed usage of sunscreen products in 2283 self-identified regular sun protection factor (SPF) consumers (RSPFC) in the United States. Subjects applied sunscreen most frequently when spending more than 3 h in the sun. Sunscreen usage peaks during the summer, with sunny weather prompting 99% usage of beach/recreational SPF products but drops to approximately 50% and 30% on partly cloudy and cloudy days, respectively, regardless of SPF product category. About half of RSPFC augment sunscreen product usage by limiting time in the sun and wearing a hat. SPF products are not reapplied by approximately 20-60% of RSPFC, depending upon product category, and reapplication was less than 33% on cloudy and partly cloudy days. Primary reasons for reapplication were water exposure, number of hours in the sun, and being active/sweating, most notably for beach/recreational SPF products. Importantly, in children, 45% of parents reported "redness" as a signal for reapplying sunscreen product. Only 10% of respondents correctly identified sunscreen products as drugs. Based on these results, while sunscreens may share common ingredients and efficacy measures, their usage by consumers varies widely depending on product type, season, weather, gender, age, and geographical location.

The bovine corneal opacity and permeability test in routine ocular irritation testing and its improvement within the limits of OECD test guideline 437.

Data on eye irritation are generally needed for the hazard identification of chemicals. As the Bovine Corneal Opacity and Permeability (BCOP) test has been accepted by many regulatory agencies for the identification of corrosive and severe ocular irritants since September 2009 (OECD Test Guideline 437, TG 437), we evaluated this alternative method for routine testing at BASF. We demonstrated our technical proficiency by testing the reference standards recommended in TG 437, and 21 additional materials with published BCOP and in vivo data. Our results matched the published in vitro data very well, but with some intentionally selected false negatives (FNs) and false positives (FPs), the concordance was 77% (24/31), with FN and FP rates of 20% (2/10) and 24% (5/21), respectively. In addition, we tested 21 in-house materials, demonstrating the utility of the BCOP assay for our own test material panel. Histopathological assessment of the corneas by light microscopy was also conducted, as this was suggested as a means of improving the identification of FNs. The histopathology corrected the classification of some FNs, but also increased the number of FPs. Parallel to the test method evaluation, we compared three new opacitometer models with the current standard device. We recommend the use of an opacitometer developed in our BASF laboratory, which has certified components and electronic data storage, resulting in what we consider to be excellent sensitivity, stability and reproducibility.

Intra- and inter-laboratory reproducibility and accuracy of the LuSens assay: A reporter gene-cell line to detect keratinocyte activation by skin sensitizers.

Several non-animal methods are now available to address the key events leading to skin sensitization as defined by the adverse outcome pathway. The KeratinoSens assay addresses the cellular event of keratinocyte activation and is a method accepted under OECD TG 442D. In this study, the results of an inter-laboratory evaluation of the "me-too" LuSens assay, a bioassay that uses a human keratinocyte cell line harboring a reporter gene construct composed of the rat antioxidant response element (ARE) of the NADPH:quinone oxidoreductase 1 gene and the luciferase gene, are described. Earlier in-house validation with 74 substances showed an accuracy of 82% in comparison to human data. When used in a battery of non-animal methods, even higher predictivity is achieved. To meet European validation criteria, a multicenter study was conducted in 5 laboratories. The study was divided into two phases, to assess 1) transferability of the method, and 2) reproducibility and accuracy. Phase I was performed by testing 8 non-coded test substances; the results showed a good transferability to naïve laboratories even without on-site training. Phase II was performed with 20 coded test substances (performance standards recommended by OECD, 2015). In this phase, the intra- and inter-laboratory reproducibility as well as accuracy of the method was evaluated. The data demonstrate a remarkable reproducibility of 100% and an accuracy of over 80% in identifying skin sensitizers, indicating a good concordance with in vivo data. These results demonstrate good transferability, reliability and accuracy of the method thereby achieving the standards necessary for use in a regulatory setting to detect skin sensitizers.

Human leukocyte elastase gene expression is regulated by PU.1 in conjunction with closely associated cytidine-rich and Myb binding sites.

Leukocyte elastase (LE) degrades connective tissue, is involved in the inflammatory process and implicated in cyclic and congenital neutropenia. The human LE gene is within a serine proteinase locus on chromosome 19 pter13.3. Our observations demonstrate that LE gene expression is regulated by PU.1, a cytidine-rich and a Myb binding site. The LE promoter has two cytidine-rich sites at -158 and -185. The -158 is the active site and it is closest to the PU.1 site. Proximity is essential to activity since separation of the -158 and PU.1 sites by a 20-base pair oligonucleotide reduced promoter activity by 50%. This suggests physical interaction between the transcription proteins binding to the PU.1 and -158 sites. The nuclear protein that binds the -158 site is present in B and T lymphocytes and an erythroleukemia cell line in addition to being abundant in the promyelocytic stage of neutrophil maturation when the LE gene is expressed. The protein binding to the -158 site is absent or expressed at low levels in non-hematopoietic cell lines. We have identified the transcription factors essential for human LE gene expression. Comparison with the mouse LE gene shows similarities and differences.

Using novel in vitro NociOcular assay based on TRPV1 channel activation for prediction of eye sting potential of baby shampoos.

The transient receptor potential vanilloid type 1 (TRPV1) channel is one of the most well-characterized pain-inducing receptors. The purpose of this study was to predict human eye stinging of 19 baby bath and shampoo formulations by studying TRPV1 activity, as measured by increase in intracellular free Ca(2+). The NociOcular test, a novel recombinant neuronal in vitro model with high expression of functional TRPV1 channels, was used to test formulations containing a variety of surfactants, preservatives, and fragrances. TRPV1-specific Ca(2+) influx was abolished when the TRPV1 channel antagonist capsazepine was applied to the cells prior to shampoo samples. The positive control, an adult shampoo that contains cocamide monoethanolamine (CMEA), a known stinging ingredient, was the most active sample tested in the NociOcular test. The negative control, a marketed baby shampoo, was negative in the NociOcular and human tests. Seven of the formulations induced stinging in the human test, and of those six were positive in the NociOcular test. Twelve formulations were classified as nonstinging in the human test, and of those ten were negative in the NociOcular test. There was no correlation between the clinical stinging results for the baby formulations and the data generated from other in vitro eye irritation assays (cytosensor microphysiometer, neutral red uptake, EpiOcular, transepithelial permeability). Our data support that the TRPV1 channel is a principal mediator of eye-stinging sensation induced by baby bath and shampoo formulations and that the NociOcular test may be a valuable in vitro tool to predict human eye-stinging sensation.

Cyclosporine A suppresses keratinocyte cell death through MPTP inhibition in a model for skin cancer in organ transplant recipients.

Transplant recipients have an elevated risk of skin cancer, with a 65- to 250-fold increase in squamous cell carcinoma. Usage of the immunosuppressant cyclosporine A (CsA) is associated with the development of skin cancer. We hypothesized that the increased incidence of skin cancer was due to the action of CsA within keratinocyte mitochondria where it can inhibit mitochondrial permeability transition pore (MPTP) opening. Normally, MPTP opening is induced by oxidative stress such as that caused by UV light and leads to cell death, thereby eliminating a cell that has been exposed to genotoxic insult. However, in the presence of CsA, damaged cells may survive and consequently form tumors. To test this hypothesis, we treated keratinocytes with levels of CsA used therapeutically in transplant patients and assessed their viability following UVA-irradiation. CsA prevented cell death by inhibiting MPTP opening, even though the levels of oxidative stress were increased markedly. Nim811, a non-immunosuppressive drug that can block the MPTP had a similar effect while the immunosuppressive drug tacrolimus that does not interact with the mitochondria had no effect. These findings suggest that CsA may promote skin cancer in transplant patients by allowing keratinocyte survival under conditions of increased genotoxic stress.

NOX4 mediates hypoxia-induced proliferation of human pulmonary artery smooth muscle cells: the role of autocrine production of transforming growth factor-{beta}1 and insulin-like growth factor binding protein-3.

Persistent hypoxia can cause pulmonary arterial hypertension that may be associated with significant remodeling of the pulmonary arteries, including smooth muscle cell proliferation and hypertrophy. We previously demonstrated that the NADPH oxidase homolog NOX4 mediates human pulmonary artery smooth muscle cell (HPASMC) proliferation by transforming growth factor-beta1 (TGF-beta1). We now show that hypoxia increases HPASMC proliferation in vitro, accompanied by increased reactive oxygen species generation and NOX4 gene expression, and is inhibited by antioxidants, the flavoenzyme inhibitor diphenyleneiodonium (DPI), and NOX4 gene silencing. HPASMC proliferation and NOX4 expression are also observed when media from hypoxic HPASMC are added to HPASMC grown in normoxic conditions, suggesting autocrine stimulation. TGF-beta1 and insulin-like growth factor binding protein-3 (IGFBP-3) are both increased in the media of hypoxic HPASMC, and increased IGFBP-3 gene expression is noted in hypoxic HPASMC. Treatment with anti-TGF-beta1 antibody attenuates NOX4 and IGFBP-3 gene expression, accumulation of IGFBP-3 protein in media, and proliferation. Inhibition of IGFBP-3 expression with small interfering RNA (siRNA) decreases NOX4 gene expression and hypoxic proliferation. Conversely, NOX4 silencing does not decrease hypoxic IGFBP-3 gene expression or secreted protein. Smad inhibition does not but the phosphatidylinositol 3-kinase (PI3K) signaling pathway inhibitor LY-294002 does inhibit NOX4 and IGFBP-3 gene expression, IGFBP-3 secretion, and cellular proliferation resulting from hypoxia. Immunoblots from hypoxic HPASMC reveal increased TGF-beta1-mediated phosphorylation of the serine/threonine kinase (Akt), consistent with hypoxia-induced activation of PI3K/Akt signaling pathways to promote proliferation. We conclude that hypoxic HPASMC produce TGF-beta1 that acts in an autocrine fashion to induce IGFBP-3 through PI3K/Akt. IGFBP-3 increases NOX4 gene expression, resulting in HPASMC proliferation. These observations add to our understanding hypoxic pulmonary vascular remodeling.

Nox4 mediates TGF-beta1-induced retinoblastoma protein phosphorylation, proliferation, and hypertrophy in human airway smooth muscle cells.

Transforming growth factor-beta1 (TGF-beta1) plays a pivotal role in increasing airway smooth muscle mass in severe asthma by inducing proliferation and hypertrophy of human airway smooth muscle. The mechanism(s) for these effects of TGF-beta1 have not been fully elucidated. In this study, we demonstrate that TGF-beta1 is a potent inducer of expression of the nonphagocyte NAD(P)H oxidase catalytic homolog Nox4, diphenylene iodonium-inhibitable reactive oxygen species production, proliferation, and hypertrophy in cultured human airway smooth muscle cells. By confocal microscopy, TGF-beta1-induced Nox4 was localized with the endoplasmic reticulum and the nucleus, implying a role for Nox4 in regulation of both the cell cycle and protein synthesis. Consistent with this hypothesis, TGF-beta1 increased retinoblastoma protein phosphorylation at both Ser807/811 and Ser780. Silencing Nox4 prevented TGF-beta1-mediated retinoblastoma protein phosphorylation, proliferation, and cell hypertrophy. TGF-beta1 also increased phosphorylation of eukaryotic translation initiation factor 4E binding protein-1 at Thr37/46, and this was likewise blocked by silencing Nox4. This is the first report to suggest a functional role for Nox4 in cell cycle transition and to demonstrate that Nox4 influences the pathobiochemistry of asthma by generating reactive oxygen species that promote TGF-beta1-induced proliferation and hypertrophy of human airway smooth muscle.

Transforming growth factor-beta1 induces Nox4 NAD(P)H oxidase and reactive oxygen species-dependent proliferation in human pulmonary artery smooth muscle cells.

Transforming growth factor-beta1 (TGF-beta1) is abundantly expressed in pulmonary hypertension, but its effect on the pulmonary circulation remains unsettled. We studied the consequences of TGF-beta1 stimulation on freshly isolated human pulmonary artery smooth muscle cells (HPASMC). TGF-beta1 initially promoted differentiation, with upregulated expression of smooth muscle contractile proteins. TGF-beta1 also induced expression of Nox4, the only NAD(P)H oxidase membrane homolog found in HPASMC, through a signaling pathway involving Smad 2/3 but not mitogen-activated protein (MAP) kinases. TGF-beta1 likewise increased production of reactive oxygen species (ROS), an effect significantly reduced by the NAD(P)H oxidase flavoprotein inhibitor diphenylene iodonium (DPI) and by Nox4 siRNAs. In the absence of TGF-beta1, Nox4 was present in freshly cultured cells but progressively lost with each passage in culture, paralleling a decrease in ROS production by HPASMC over time. At a later time point (72 h), TGF-beta1 promoted HPASMC proliferation in a manner partially inhibited by Nox4 small interfering RNA and dominant negative Smad 2/3, indicating that TGF-beta1 stimulates HPASMC growth in part by a redox-dependent mechanism mediated through induction of Nox4. HPASMC activation of the MAP kinases ERK1/2 was reduced by the NAD(P)H oxidase inhibitors DPI and 4-(2-aminoethyl)benzenesulfonyl fluoride, suggesting that TGF-beta1 may facilitate proliferation by upregulating Nox4 and ROS production, with transient oxidative inactivation of phosphatases and augmentation of growth signaling cascades. These findings suggest that Nox4 is the relevant Nox homolog in HPASMC. This is the first observation that TGF-beta1 regulates Nox4, with important implications for mechanisms of pulmonary vascular remodeling.