Brad L. Upham

Single-walled carbon nanotubes dispersed in aqueous media via non-covalent functionalization: Effect of dispersant on the stability, cytotoxicity, and epigenetic toxicity of nanotube suspensions

As the range of applications for carbon nanotubes (CNTs) rapidly expands, understanding the effect of CNTs on prokaryotic and eukaryotic cell systems has become an important research priority, especially in light of recent reports of the facile dispersion of CNTs in a variety of aqueous systems including natural water. In this study, single-walled carbon nanotubes (SWCNTs) were dispersed in water using a range of natural (gum arabic, amylose, Suwannee River natural organic matter) and synthetic (polyvinyl pyrrolidone, Triton X-100) dispersing agents (dispersants) that attach to the CNT surface non-covalently via different physiosorption mechanisms. The charge and the average effective hydrodynamic diameter of suspended SWCNTs as well as the concentration of exfoliated SWCNTs in the dispersion were found to remain relatively stable over a period of 4 weeks. The cytotoxicity of suspended SWCNTs was assessed as a function of dispersant type and exposure time (up to 48 h) using general viability bioassay with Escherichia coli and using neutral red dye uptake (NDU) bioassay with WB-F344 rat liver epithelia cells. In the E. coli viability bioassays, three types of growth media with different organic loadings and salt contents were evaluated. When the dispersant itself was non-toxic, no losses of E. coli and WB-F344 viability were observed. The cell viability was affected only by SWCNTs dispersed using Triton X-100, which was cytotoxic in SWCNT-free (control) solution. The epigenetic toxicity of dispersed CNTs was evaluated using gap junction intercellular communication (GJIC) bioassay applied to WB-F344 rat liver epithelial cells. With all SWCNT suspensions except those where SWCNTs were dispersed using Triton X-100 (wherein GJIC could not be measured because the sample was cytotoxic), no inhibition of GJIC in the presence of SWCNTs was observed. These results suggest a strong dependence of the toxicity of SWCNT suspensions on the toxicity of the dispersant and point to the potential of non-covalent functionalization with non-toxic dispersants as a method for the preparation of stable aqueous suspensions of biocompatible CNTs.

Inhibition of gap junctional intercellular communication by perfluorinated fatty acids is dependent on the chain length of the fluorinated tail

Perfluorinated fatty acids (PFFAs), such as perfluorooctanoic acid (PFOA) and perfluorodecanoic acid (PFDA), are known peroxisome proliferators and hepatocarcinogens. A causal link between an increase in the oxidative stress by peroxisomes and tumor promotion has been proposed to explain the hepatocarcinogenicity of PFOA and PFDA. However, the down‐regulation of gap junctional intercellular communication (GJIC) has also been linked to the tumor‐promoting properties of many carcinogens. Therefore, the effect of PFFAs on GJIC in WB‐rat liver epithelial cells was determined. The chain length of the PFFAs tested for an effect on GJIC ranged from 2 to 10, 16 and 18 carbons. Carbon lengths of 7 to 10 inhibited GJIC in a dose–response fashion, whereas carbon lengths of 2 to 5, 16 and 18 did not appreciably inhibit GJIC. Inhibition occurred within 15 min and was reversible, with total recovery from inhibition occurring within 30 min after the removal of the compound from the growth medium. This short time of inhibition suggests that GJIC was modified at the post‐translational level. Also, this short time period was not long enough for peroxisome proliferation. The post‐translational modification of the gap junction proteins was not a consequence of altered phosphorylation as determined by Western blot analysis. Perfluorooctanesulfonic acid also inhibited GJIC in a dose–response fashion similar to PFDA, indicating that the determining factor of inhibition was probably the fluorinated tail, which required 7–10 carbons. Our results suggest that PFFAs could potentially act as hepatocarcinogens at the level of gap junctions in addition to or instead of through peroxisome proliferation.Int. J. Cancer 78:491–495, 1998.

Ignored Hallmarks of Carcinogenesis: Stem Cells and Cell‐Cell Communication

Abstract: Hanahan and Weinberg (2000, Cell 100: 57‐70) listed “hallmarks” of cancer that must be considered in order to understand the underlying determinants of carcinogenesis: (a) self‐sufficiency in growth signals; (b) insensitivity to growth‐inhibitory (antigrowth) signals; (c) evasion of programmed cell death (apoptosis); (d) limitedless replicative potential; (e) sustained angiogenesis; and (f) tissue invasion and metastasis. While these are important phenotypic markers, important concepts—the role of pluripotent stem cells and gap junctional intercellular communication (GJIC)—must be brought into this analysis of carcinogenesis. Carcinogenesis is a multistage, multimechanism process consisting of a single cell that has been irreversibly blocked from terminal differentiation (the initiation stage). The promotion phase is a potentially reversible or interruptible clonal expansion of the initiated cell by a combination of growth stimulation and inhibition of apoptosis. When the expanded initiated cells accrue sufficient mutations and epigenetic alterations to become growth stimulus independent and resistant to growth inhibitors and apoptosis, to have unlimited replicative potential and invasive and metastatic phenotypes, then the progression phase has been achieved. The hypothesis that integrates these hallmarks is that the stem cell and its early progenitor cell are the target cells for the initiation event. These cells are naturally immortal and become mortal only when they are induced to terminally differentiate and lose their telomerase activity. These two types of initiated cells are suppressed by either secreted negative growth regulators (the stem cells) or GJIC (the early initiated progenitor cells). Promoters inhibit either the secreted growth inhibitor to initiated stem cells or GJIC between the initiated progenitor cells and the normal progenitor cells. When a stable resistance to the secreted negative growth regulator or permanent downregulation of GJIC has occurred, the cell has entered the progression phase. These two new concepts contradict the current paradigm that the first phase of carcinogenesis is the immortalization of a normal cell followed by its neoplastic transformation. Our hypothesis is that the first stage of carcinogenesis must prevent the “mortalization” or terminal differentiation of a naturally immortal cell. Chemoprevention and chemotherapeutic implications suggest that one must induce connexin genes in initiated stem cells and restore GJIC in initiated early progenitor cells.

Oxidative-Dependent Integration of Signal Transduction with Intercellular Gap Junctional Communication in the Control of Gene Expression

Research on oxidative stress focused primarily on determining how reactive oxygen species (ROS) damage cells by indiscriminate reactions with their macromolecular machinery, particularly lipids, proteins, and DNA. However, many chronic diseases are not always a consequence of tissue necrosis, DNA, or protein damage, but rather to altered gene expression. Gene expression is highly regulated by the coordination of cell signaling systems that maintain tissue homeostasis. Therefore, much research has shifted to the understanding of how ROS reversibly control gene expression through cell signaling mechanisms. However, most research has focused on redox regulation of signal transduction within a cell, but we introduce a more comprehensive-systems biology approach to understanding oxidative signaling that includes gap junctional intercellular communication, which plays a role in coordinating gene expression between cells of a tissue needed to maintain tissue homeostasis. We propose a hypothesis that gap junctions are critical in modulating the levels of second messengers, such as low molecular weight reactive oxygen, needed in the transduction of an external signal to the nucleus in the expression of genes. Thus, any comprehensive-systems biology approach to understanding oxidative signaling must also include gap junctions, in which aberrant gap junctions have been clearly implicated in many human diseases.

Determination of the epigenetic effects of ochratoxin in a human kidney and a rat liver epithelial cell line.

Epidemiological studies have implicated ochratoxin A (OTA), a fungal metabolic-contaminant of animal and human food sources, in Balkan Endemic Nephropathy and renal tumors. Many environmental toxicants operate through nongenotoxic mechanisms that epigenetically control gene expression leading to a diseased state. Gap junctional intercellular communication (GJIC) plays a central role in the epigenetic control of genes in which alteration of normal GJIC has been implicated in many human pathologies, including cancer, teratogenesis, reproductive dysfunction and peripheral neuropathies. The cell proliferative stages of human diseases, such as cancer, also involves the induction of signal transduction pathways controlling the mitogenic steps, in which the mitogen activated protein kinases (MAPK), such as extracellular receptor kinase (ERK) and p38, are central to mitogenesis. We therefore determined the effects of OTA on GJIC and MAPK in a human kidney and rat liver epithelial cell line. OTA reversibly inhibited GJIC at noncytotoxic doses in the rat liver but not the human kidney cell line. Similarly, OTA was also a strong activator of MAPK, ERK and p38, in the rat liver cells but only weakly activated ERK and had no affect on p38 in the human kidney cell line. Another hallmark of human diseases is an abnormal alteration of apoptosis, also known as programmed cell death. We used our myc-transfected cell line, which exhibits higher levels of apoptosis, to test the effects of OTA on apoptosis. OTA greatly induced apoptosis in this cell line, which is contrary to the effects of most tumor promoters. In summary, OTA exhibits tumor promoting properties in the liver, but the effects of OTA on the human kidney epithelial cells suggested a lack of tumorigenic activity assuming that these epithelial cells, like the rat liver epithelial cells, are a primary target for carcinogens. These results also indicate that the nephrotoxicity of OTA either does not involve GJIC, assuming these epithelial cells play a vital role in kidney physiology, or that a more differentiated kidney cell type is the target for OTA toxicity, of which the role of GJIC remains unknown.

Inhibitory effect of pentachlorophenol on gap junctional intercellular communication in rat liver epithelial cells in vitro

To understand the initiating/promoting actions of pentachlorophenol (PCP), a non-mutagenic hepatocarcinogen, and its metabolite, tetrachlorohydroquinone (TCHQ), we investigated the effects of each chemical on gap junctional intercellular communication (GJIC) in rat liver epithelial cells (WB cells) by the scrape-loading and dye transfer method. After treatment with PCP, the GJIC was initially inhibited at 4 h but was restored in 6-8 h, followed by a second phase of inhibition between 16 and 24 h. Both the first and second inhibitions were concentration-dependent and were restored by 2-4 h after removal of PCP. The phosphorylation state of connexin 43 (CX43) and its localization on the plasma membrane were unchanged up to 24 h after treatment; however, this was accompanied by a decrease in the CX43 protein level. No inhibitory effect was apparent on the GJIC of cells treated with TCHQ. These results suggest that PCP may play a critical role of promoting activity via non-mutagenic mechanisms.

Structure-Activity–Dependent Regulation of Cell Communication by Perfluorinated Fatty Acids using in Vivo and in Vitro Model Systems

Background Perfluoroalkanoates, [e.g., perfluorooctanoate (PFOA)], are known peroxisome proliferators that induce hepatomegaly and hepatocarcinogenesis in rodents, and are classic non-genotoxic carcinogens that inhibit in vitro gap-junctional intercellular communication (GJIC). This inhibition of GJIC is known to be a function of perfluorinated carbon lengths ranging from 7 to 10. Objectives The aim of this study was to determine if the inhibition of GJIC by PFOA but not perfluoropentanoate (PFPeA) observed in F344 rat liver cells in vitro also occurs in F344 rats in vivo and to determine mechanisms of PFOA dysregulation of GJIC using in vitro assay systems. Methods We used an incision load/dye transfer technique to assess GJIC in livers of rats exposed to PFOA and PFPeA. We used in vitro assays with inhibitors of cell signaling enzymes and antioxidants known to regulate GJIC to identify which enzymes regulated PFOA-induced inhibition of GJIC. Results PFOA inhibited GJIC and induced hepatomegaly in rat livers, whereas PFPeA had no effect on either end point. Serum biochemistry of liver enzymes indicated no cytotoxic response to these compounds. In vitro analysis of mitogen-activated protein kinase (MAPK) indicated that PFOA, but not PFPeA, can activate the extracellular receptor kinase (ERK). Inhibition of GJIC, in vitro, by PFOA depended on the activation of both ERK and phosphatidylcholine-specific phospholipase C (PC-PLC) in the dysregulation of GJIC in an oxidative-dependent mechanism. Conclusions The in vitro analysis of GJIC, an epigenetic marker of tumor promoters, can also predict the in vivo activity of PFOA, which dysregulated GJIC via ERK and PC-PLC.

Oxidative stress in liver and brain of the hatchling chicken (Gallus domesticus) following in ovo injection with TCDD

2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) was injected into chicken eggs prior to incubation to study possible mechanisms of toxicity and teratogenicity. One of the suggested mechanisms of teratogenicity is oxidative stress. Eggs were injected simultaneously with TCDD and cotreatment compounds in an attempt to prevent oxidative stress or to block cytochrome P450 activity. Indicators of oxidative stress were assessed in livers and brains of hatchling chicks. In ovo, exposure to TCDD caused significant effects on indicators of oxidative stress in liver, but not in the brain of the hatchling chicks. TCDD did not significantly affect superoxide production. In liver, TCDD treatment caused a decrease in glutathione content and glutathione peroxidase activity and an increase in the ratio of oxidized to reduced glutathione. TCDD increased the susceptibility to lipid peroxidation and oxidative DNA damage in liver. Administration of the antioxidants vitamin E and vitamin A provided partial protection against TCDD-induced oxidative stress in liver. The lack of effect of TCDD in chicken brain could be due to the low cytochrome P4501A activity in this tissue and little accumulation of TCDD in brain compared to liver. Phenytoin, a known inducer of oxidative stress, caused a decrease in glutathione content and an increase in susceptibility to lipid peroxidation in both liver and brain and increased oxidative DNA damage in brain. Responsiveness varied among individual animals, but measures of the oxidative stress were correlated.

Reduced gap junctional intercellular communication and altered biological effects in mouse osteoblast and rat liver oval cell lines transfected with dominant‐negative connexin 43

Gap junctional intercellular communication (GJIC) maintains normal growth and differentiation of cells in a tissue. The intercellular molecules traversing gap junctions are largely unknown, but the molecular weight (MW) cutoff is normally 1200 Da. No differences in dye transfer were observed in normal or vector controls of WB‐F344 rat liver epithelial or mouse osteoblastic MC3T3‐E1 cells with either Lucifer Yellow (LY) with a MW of 457 Da (LY‐457) or LY with a MW of 649 Da (LY‐649). Transfection of a dominant negative‐connexin 43 (Cx43) gene decreased GJIC (>50%) when LY‐649 was used, however, normal GJIC was observed in both cell lines when LY‐457 was used. Therefore, the MW cut off in these clones was considerably less than the wild type. The dominant negative clones of the MC3T3‐E1 cells exhibited over 90% less alkaline phosphatase (ALPase) activity and calcium deposition after the induction of differentiation. Similarly, dominant negative Cx43 inhibited gene expression of ALPase and bone sialoprotein but not osteocalcin in MC3T3‐E1. WB‐F344 cells normally exhibit a biphasic response to 12‐O‐tetradecanoylphorbol‐13‐acetate (TPA) where inhibition of GJIC recovers after 2 h, but the dominant negative clones showed no recovery from inhibition of GJIC by TPA. Dominant negative Cx43 also inhibited the formation of network‐like structures by WB‐F344 cells on Matrigel. These results demonstrate that the dominant negative gene transfected into cell types containing the wild‐type connexins result in diminished channel sizes, thus allowing the determination of whether distinct biological endpoints, i.e., differentiation, are dependent upon either small or high MW intercellular signals.

Tumor promoting properties of a cigarette smoke prevalent polycyclic aromatic hydrocarbon as indicated by the inhibition of gap junctional intercellular communication via phosphatidylcholine-specific phospholipase C

Inhibition of gap junctional intercellular communication (GJIC) and the activation of intracellular mitogenic pathways are common hallmarks of epithelial derived cancer cells. We previously determined that the 1‐methyl and not the 2‐methyl isomer of anthracene, which are prominent cigarette smoke components, activated extracellular receptor kinase, and inhibited GJIC in WB‐F344 rat liver epithelial cells. Using these same cells, we show that an immediate upstream response to 1‐methylanthracene was a rapid (<1 min) release of arachidonic acid. Inhibition of phosphatidylcholine‐specific phospholipase C prevented the inhibition of GJIC by 1‐methylanthracene. In contrast, inhibition of phosphatidylinositol specific phospholipase C, phospholipase A2, diacylglycerol lipase, phospholipase D, protein kinase C, and tyrosine protein kinases had no effect on 1‐methylanthracene‐induced inhibition of GJIC. Inhibition of protein kinase A also prevented inhibition of GJIC by 1‐methylanthracene. Direct measurement of phosphatidylcholine‐specific phospholipase C and sphingomyelinase indicated that only phosphatidylcholine‐specific phospholipase C was activated in response to 1‐methylanthracene, while 2‐methylanthracene had no effect. 1‐methylanthracene also activated p38‐mitogen activated protein kinase; however, like extracellular kinase, its activation was not involved in 1‐methylanthracene‐induced regulation of GJIC, and this activation was independent of phosphatidylcholine‐specific phospholipase C. Although mitogen activated protein kinases were activated, Western blot analyzes indicated no change in connexin43 phosphorylation status. Our results indicate that phosphatidylcholine‐specific phospholipase C is an important enzyme in the induction of a tumorigenic phenotype, namely the inhibition of GJIC; whereas mitogen activated protein kinases triggered in response to 1‐methylanthracene, were not involved in the deregulation of GJIC. (Cancer Sci 2008; 99: 696–705)