Geochemical characterization and renal cell toxicity of water-soluble extracts from U.S. Gulf Coast lignite.

An assortment of organic material can leach from lignite (low-rank coal) in water, and the water-soluble fraction from lignite has been associated with adverse health effects in areas of the Balkans. Recent efforts have been made to evaluate this hypothesis in other areas where lignite is in contact with groundwater like in the U.S. Gulf Coast region. In this study, five Gulf Coast lignite samples were extracted with water, and the water-soluble portion of the coal was then characterized by total organic carbon, UV-Vis spectroscopy, and gas chromatography/mass spectrometry. Additionally, human kidney cells (HK-2) were exposed to water-soluble extracts of Gulf Coast lignite to assess toxicity. Cell viability was measured, and a dose-response curve was used to generate IC50 values that ranged from 490 to 3000 ppm. The most toxic extract (Dolet Hills) was from Louisiana where lignite-derived organic material has been previously linked to high incidence of renal pelvic cancer. Concentrations of nephrotoxic metals (As, Cd, Co, Cu, Hg, Pb, V, Zn) were screened and were below those considered toxic to renal cells. We conclude that leachates from lignite do indeed have toxic affects on cultured human renal cells. Although the IC50 values are higher than the concentration of organic matter in the local groundwater, typically < 5 ppm, the effects of long-term low-level exposure is not known.

Interleukin (IL)-6 modulates transforming growth factor-beta expression in skin and dermal fibroblasts from IL-6-deficient mice.

BACKGROUND: Interleukin (IL-6) and transforming growth factor (TGF)-beta have been shown to play a role in skin development and maintenance. OBJECTIVES: A link between these two cytokines has yet to be identified and therefore in this study we investigated the modulation of TGF-beta1 and TGF-beta type 2 receptor (TGF-betaR2) by IL-6 in skin. METHODS: An IL-6 knockout (IL-6KO) fibroblast-populated lattice model and intradermal injections of IL-6 into unwounded IL-6KO mice were used to investigate the direct effects of IL-6 treatment on TGF-beta and TGF-betaR2 expression and to determine the signalling mechanism. In addition, IL-6KO and C57BL/6 control mice were wounded by a 4-mm punch biopsy to monitor expression of TGF-beta1 and TGF-betaR2 within a wound over time. The expression of TGF-beta1 and TGF-betaR2 was assessed by real-time quantitative polymerase chain reaction, enzyme-linked immunosorbent assay and immunohistology. RESULTS: Recombinant IL-6 treatment of IL-6KO lattices and intradermal injections of IL-6 showed a significant induction of TGF-beta1 mRNA and protein, with TGF-beta1 expression localized in the dermis, while TGF-betaR2 expression was primarily in the epidermis in IL-6KO mice. During healing, the expression of TGF-beta1 and TGF-betaR2 mRNA was significantly greater in unwounded and 7-day-old wounds from wild-type mice; however, protein expression did not differ. Treatment with signal transduction inhibitors indicated that IL-6 modulates TGF-beta through a mitogen-activated protein kinase/extracellular signal-regulated kinase (Mapk/Erk)-dependent mechanism. CONCLUSION: These studies indicate that IL-6 has the ability to modulate the expression of TGF-beta and TGF-betaR2 to varying degrees in the skin, which may provide a possible mechanism for defining the role of IL-6 in skin maintenance and a new association of IL-6 with TGF-beta in pathologies associated with fibrosis.

Interleukin-6 (IL-6) modulates migration and matrix metalloproteinase function in dermal fibroblasts from IL-6KO mice.

BACKGROUND: Interleukin-6-deficient (IL-6KO) mice display significantly delayed cutaneous wound healing characterized by decreased re-epithelialization, granulation tissue and wound closure. Dermal fibroblasts are one of the principal cell types found in granulation tissue and mediate numerous processes during healing. OBJECTIVES: To investigate the effects that IL-6 might have on granulation tissue formation and fibroblast motility. As fibroblast motility is associated with matrix metalloproteinase (MMP) activity, the expression of MMP-2 and the tissue inhibitors of metalloproteinase (TIMP)-1 and -2 were assessed. METHODS: Punch biopsies (4 mm) were performed in the skin of IL-6KO and C57BL/6 mice. The expression of MMP-2, TIMP-1 and -2 in wound tissue was monitored over time. Cellular infiltration and granulation tissue formation was monitored by subcutaneous implantation of polyvinyl alcohol (PVA) sponges. A free-floating collagen lattice model was also used to investigate the direct effects of IL-6 treatment on isolated IL-6KO fibroblasts. The expression of MMP-2, and the inhibitors TIMP-1 and -2, were assessed via real-time quantitative polymerase chain reaction and enzyme-linked immunosorbent assay. RESULTS: IL-6KO wounds showed impaired granulation tissue formation 5 days postwounding and fewer fibroblasts had populated the PVA matrices 7 days after implantation in IL-6KO mice compared with wild-type C57BL/6. The mRNA and protein expression of MMP-2 and TIMP-2 mRNA was increased in IL-6KO mice compared with wild-type mice beyond 1 day postwounding, while the expression of TIMP-1 mRNA was transiently higher in IL-6KO only 3 days postwounding. Treatment of collagen lattices with various concentrations of rmIL-6 again showed a dose-response decrease in mRNA and protein expression of MMP-2 and TIMP-2 protein expression, compared with saline control, while TIMP-1 did not appear to be significantly modulated. CONCLUSIONS: These results indicate that IL-6 influences the function of fibroblasts in wounds, and one mechanism of this regulation may be through the modulation of MMP-2 and TIMP proteins.

Stress induced in heart and other tissues by rat dermal exposure to JP-8 fuel.

Limited information is available regarding the development of systemic organ stress by dermal exposure to JP-8 fuel. In this study, the systemic stress potential of this fuel is evaluated in a rat model subjected to dermal applications of JP-8 for 7 days at 300 microl per day. Tissue histology indicated that JP-8 induces morphological alterations that suggest that tissue stress in the heart is more substantial than stress in the kidney and liver. Immunoblot analysis of tissues revealed increased levels of the inducible heat shock protein 70 (HSP70) in the heart, kidney, and liver after this dermal JP-8 exposure. This exposure also leads to increased levels of heme oxygenase-1 (HO-1/HSP3) in the liver. Additionally during this exposure, a negative regulator of inflammation, IkappaBalpha (inhibitor of NF-kappaB), was increased in the liver, slightly increased in the kidney, and not increased in the heart. Two regions of the rat brain were also examined and HSP70 and IkappaBalpha were increased in the cerebellum but not significantly increased in the cortex. This study indicates dermal JP-8 exposure causes systemic alterations that are associated with cytoprotective activities (e.g., in the liver) as well as potentially toxic mechanisms (heart and kidney).

The role of tumor necrosis factor-alpha in liver toxicity, inflammation, and fibrosis induced by carbon tetrachloride.

Hepatic expression of the proinflammatory cytokine tumor necrosis factor-alpha (TNFalpha) occurs in many acute and chronic liver diseases, as well as following exposure to hepatotoxic chemicals, and is believed to help influence both the damage and repair processes that occur following these insults by regulating additional mediators. We examined the role of TNFalpha in transgenic mice deficient in TNF receptors (TNFR) utilizing carbon tetrachloride (CCl(4)) as a model hepatotoxic agent that allowed for the evaluation of necrosis, inflammation, and fibrosis. Hepatocyte damage, as evident by local areas of liver necrosis and elevated levels of serum transaminase, occurred to a similar degree in wild-type and TNFR-deficient knockout (KO) mice following acute exposure to CCl(4). In contrast, the inflammatory response, manifested as an inflammatory cell influx, as well as induction of chemokines and adhesion molecules that occurred in wild-type mice following treatment with CCl(4), was not as evident in TNFR-KO mice. This response was associated primarily with type-1 (TNFR1) rather than type-2 (TNFR2) receptor responses. Liver fibrosis resulting from chronic CCl(4) exposure was also markedly dependent upon TNFalpha as demonstrated by almost a complete histological absence of fibrosis in TNFR-deficient mice. This was further supported by marked reductions in procollagen and transforming growth factor beta synthesis in TNFR-deficient mice. Taken together, these results indicate that TNFalpha is responsible for regulating products that induce inflammation and fibrosis but not direct hepatocyte damage in CCl(4)-induced hepatotoxicity.

Regulation and role of interleukin 6 in wounded human epithelial keratinocytes.

Dermal wounding is accompanied by inflammation and the resulting proinflammatory cytokines, including interleukin (IL)-6, are thought to play an important role in the repair process. IL-6 is produced by normal human keratinocytes to various dermatological diseases and we have recently shown it is also required for normal wound repair. However, neither the events responsible for its induction nor its role in repair have been clearly identified. Using a recently developed in vitro wounding model, we demonstrate that IL-6 mRNA is expressed and immunoreactive IL-6 is released from cultures of human epidermal keratinocytes (NHEKs) following wounding. The transcription factors, NF kappa B and NF-IL-6 (C/EBP beta), which coordinately help regulate IL-6 expression, were activated following wounding and preceded the appearance of IL-6. Addition of IL-1 alpha to NHEK cultures increased IL-6 production and activated NF kappa B and C/EBP beta. Addition of the IL-1 alpha receptor antagonist inhibited both IL-6 mRNA expression and the transcription factors following wounding. Immunoreactive IL-1 alpha was detected in the medium following wounding in the absence of new message. Furthermore, addition of IL-6 to NHEK cultures decreased the expression of keratins 1 and 10, differentiation markers of keratinocytes, while proliferation was not affected. Taken together, these data indicate that constitutive keratinocyte-derived IL-1 alpha is a stimulus for IL-6 production in wounded epidermis, the response involves NF kappa B and C/EBP beta transcription factors, and IL-6 may be associated with modulation of keratinocyte differentiation rather than proliferation.

Interleukin-6 treatment augments cutaneous wound healing in immunosuppressed mice.

It has been postulated that the inflammatory response that occurs after cutaneous wounding is a prerequisite for healing and that inflammatory cytokines, such as interleukin-6 (IL-6) are involved in this process. We showed previously that IL-6-deficient mice display delayed wound healing, which could be reversed by administration of a murine IL-6 expression plasmid or recombinant murine IL-6 (rMuIL-6). In the present study, we observed that delayed cutaneous wound healing, which occurs as a result of glucocorticoid-induced immunosuppression, can also be reversed by rMuIL-6, as evidenced by epithelialization, granulation tissue formation, and wound closure. In vehicle control mice, rMuIL-6 did not augment healing but rather delayed the process. Immunochemical studies indicated that the expression of matrix metalloproteinase-10 (MMP-10) was increased in dexamethasone-treated mice and that rMuIL-6 treatment reduced its expression, indicating that IL-6 may influence dermal matrix formation and, specifically, collagen synthesis. These results demonstrate that IL-6 can restore abnormal wound repair that occurs in immunodeficiency and suggest its use as a potential therapy.

Role of inflammation in chemical-induced hepatotoxicity.

The liver, which is the major organ responsible for the metabolism of drugs and toxic chemicals, is also the primary target organ for many toxic chemicals. Increasing evidence has indicated that inflammatory processes are intimately involved in chemical-induced hepatotoxic processes, and like other inflammatory diseases, such as autoimmunity, are responsible for producing mediators that can effect liver damage or repair. This review will summarize our current understanding of how inflammatory processes influence hepatic pathology and repair following exposure to established hepatotoxic chemicals including carbon tetrachloride, an industrial chemical, and acetaminophen, a widely used analgesic.

Impaired cutaneous wound healing in interleukin-6-deficient and immunosuppressed mice.

It has been postulated that an inflammatory response after cutaneous wounding is a prerequisite for healing, and inflammatory cytokines, such as interleukin-6 (IL-6), might be intimately involved in this process. IL-6-deficient transgenic mice (IL-6 KO) displayed significantly delayed cutaneous wound healing compared with wild-type control animals, requiring up to threefold longer to heal. This was characterized by minimal epithelial bridge formation, decreased inflammation, and granulation tissue formation. Using electrophoretic mobility shift assays of wound tissue from IL-6 KO mice, decreased AP-1 transcription factor activation was shown compared with wild-type mice 16 h after wounding. In situ hybridization of wound tissue from wild-type mice revealed IL-6 mRNA expression primarily in the epidermis at the leading edge of the wound. Delayed wound healing in IL-6 KO mice was reversed with a single dose of recombinant murine IL-6 or intradermal injection of an expression plasmid containing the full-length murine IL-6 cDNA. Treatment with rmIL-6 also reconstituted wound healing in dexamethasone-treated immunosuppressed mice. The results of this study may indicate a potential use for IL-6 therapeutically where cutaneous wound healing is impaired.

The role of tumor necrosis factor alpha in chemical-induced hepatotoxicity.

Only recently have toxicologists come to understand the role of inflammation, and TNFalpha specifically, in classical toxicological processes. This relationship appears fairly complex, as inflammation and proliferation may well be only one facet of a time- and dose-dependent continuum of toxicological and repair processes. Not surprisingly, considerable efforts are being undertaken using our newly found understanding of molecular control to develop specific and safe chemical, biological, and molecular regulators of TNFalpha for potential therapeutic use. Their effectiveness in controlling environmental or occupational diseases has yet to be established.

The modulation of B16BL6 melanoma metastasis is not directly mediated by cytolytic activity of natural killer cells in alcohol-consuming mice.

BACKGROUND: Previous studies in our laboratory indicate that alcohol consumption suppresses the metastasis of B16BL6 melanoma, whereas the cytolytic activity of natural killer (NK) cells is decreased in female C57BL/6 mice given 20% w/v alcohol in their drinking water. In the present study, we further evaluated the involvement of NK cells and alcohol consumption in the cytolytic activity of NK cells, the surface expression of NK phenotypic markers, and metastasis of B16BL6 melanoma in C57BL/6 beige (bgJ/bgJ) mutant mice, which possess inherently low NK-cell cytolytic activity. METHODS: Beige and control (bgJ/+) mice were given either water or 20% w/v of alcohol in drinking water for 6 1/2 to 7 weeks before assay for cytolytic activity, surface marker expression, and inoculation with B16BL6 melanoma intravenously or into the pinna of the ear. RESULTS: NK cytolytic activity was suppressed in beige mice, and alcohol consumption did not modulate further the cytolytic activity. Beige mice had a lower percentage of NK cells in the peripheral blood and spleen than control mice. Peripheral blood lymphocytes from beige mice also exhibited a reduced percentage of CD4+ T lymphocytes. Alcohol consumption similarly reduced the percentages of NK1.1- and LGL-1-expressing lymphocytes in the peripheral blood and spleen and reduced the percentage of CD8+ T lymphocytes in the peripheral blood in both control and beige mice. Tumor lung colonization was increased in beige mice relative to control mice after intravenous inoculation of B16BL6 melanoma. The increase was more pronounced in water-drinking beige mice than in control mice irrespective of alcohol consumption. Tumor lung colonization was significantly decreased (p < 0.05) by alcohol consumption in one experiment and partially decreased (p = 0.07) in the other. Mice that were inoculated into the pinna of the ear also exhibited a blunted antimetastatic response to alcohol consumption. CONCLUSIONS: These data suggest that the presence of the beige mutation diminishes the antimetastatic effect of alcohol consumption and that there is no interaction between alcohol consumption and NK-cell activity in the modulation of lung metastasis of B16BL6 melanoma cells.

TNF-alpha regulates transforming growth factor-alpha expression in regenerating murine liver and isolated hepatocytes.

TNF-alpha is a pleotropic proinflammatory cytokine that has been implicated as a contributing factor in a number of disease processes, primarily through its ability to induce the expression of inflammatory and cytotoxic mediators. TNF-alpha is also involved in cell growth accompanying the healing process in multiple organ systems and influences liver repair following hepatotoxic damage or regeneration following partial hepatectomy. In this respect, TNF-alpha is a known mitogen for hepatocytes. In this paper we describe a novel role for TNF-alpha in the modulation of expression of TGF-alpha, the latter being a complete hepatocyte mitogen. TNF-alpha directly up-regulates TGF-alpha mRNA by up to 7-fold in isolated mouse hepatocytes, whereas neutralization of TNF-alpha significantly decreased liver mRNA and protein expression of TGF-alpha following chemical-induced hepatotoxicity. That TNF-alpha directly stimulated TGF-alpha was suggested by the inability of either anti-IL-6 Abs or cycloheximide to inhibit TNF-alpha-induced TGF-alpha expression in hepatocytes. However, in the presence of anti-TGF-alpha neutralizing Abs, the mitogenic activity of TNF-alpha is abrogated. Using cells transfected with the TGF-alpha promoter, and an RNA polymerase inhibitor, it was shown that TNF-alpha modulates TGF-alpha expression through both pre- and posttranscriptional events. Taken together, these data suggest that TNF-alpha participates in liver repair and regeneration, in part, by directly inducing the expression of TGF-alpha.

Immunotoxicology: role of inflammation in chemical-induced hepatotoxicity.

The liver, which is the major organ responsible for the metabolism of drugs and chemicals, is also the primary target organ for many toxic chemicals. Increasing evidence has indicated that inflammatory processes are intimately involved in chemical-induced hepatotoxic processes, and like other inflammatory diseases, such as autoimmunity, are responsible for producing mediators which can effect liver damage or repair. This review will summarize the authors' current understanding of how inflammatory processes influence hepatic pathology and repair following exposure to established hepatotoxic chemicals including carbon tetrachloride (CCl4), an industrial chemical, and acetaminophen (APAP), a widely used analgesic.

Essential role of tumor necrosis factor alpha in alcohol-induced liver injury in mice.

BACKGROUND & AIMS: Tumor necrosis factor (TNF)-alpha is associated with increased mortality in alcoholics, but its role in early alcohol-induced liver injury is not fully understood. Recently, it was shown that injury induced by the enteral alcohol delivery model of Tsukamoto and French was reduced by antibodies to TNF-alpha. To obtain clear evidence for or against the hypothesis that TNF-alpha is involved, we studied TNF receptor 1 (TNF-R1, p55) or 2 (TNF-R2, p75) knockout mice. METHODS: Long-term enteral alcohol delivery was modified for male gene-targeted mice lacking TNF-R1 and TNF-R2. Animals were given a high-fat liquid diet continuously with either ethanol or isocaloric maltose-dextrin as a control for 4 weeks. RESULTS: Ethanol elevated serum levels of alanine aminotransferase nearly 3-fold in wild-type and TNF-R2 knockout mice but not in TNF-R1 knockout mice. Likewise, ethanol caused severe liver injury in wild-type mice (pathology score, 5.5 +/- 0.6) and TNF-R2 knockout mice (pathology score, 5.0 +/- 0.4), but not in TNF-R1 knockout mice (pathology score, 0.8 +/- 0.4; P < 0.001). CONCLUSIONS: Long-term ethanol feeding caused liver injury in wild-type and TNF-R2 knockout mice but not in TNF-R1 knockout mice, providing solid evidence in support of the hypothesis that TNF-alpha plays an important role in the development of early alcohol-induced liver injury via the TNF-R1 pathway. Moreover, the long-term enteral ethanol feeding technique we described for the first time for knockout mice provides a useful new tool for alcohol research.

Functional asymmetries in the quality of handwriting movements: a kinematic analysis.

Writing hand preference is a prominent functional asymmetry, but biomechanical factors may also contribute to any kinematic differences in the quality of handwriting movements performed by either hand. Eighteen dextral participants used a noninking pen with their right or left hand to write cursive letter ls, inverted ls, and their mirror images (to control for biomechanical differences) on a graphics tablet. Kinematic analysis of the scaling, consistency, efficiency, and shape of writing stroke trajectories revealed functional asymmetries between hands. The right hand was faster and produced more efficient strokes, which were of more consistent length, duration, and peak velocity. Differences between hands do not simply reflect biomechanical factors; therefore, the documentation of any functional asymmetries may allow their subsequent use as markers of underlying pathology in conditions such as schizophrenia.

Response (re-)programming in aging: a kinematic analysis.

BACKGROUND: Age-related motor slowing may reflect either motor programming deficits, poorer movement execution, or mere strategic preferences for online guidance of movement. We controlled such preferences, limiting the extent to which movements could be programmed. METHODS: Twenty-four young and 24 older adults performed a line drawing task that allowed movements to be prepared in advance in one case (i.e., cue initially available indicating target location) and not in another (i.e., no cue initially available as to target location). Participants connected large or small targets illuminated by light-emitting diodes upon a graphics tablet that sampled pen tip position at 200 Hz. RESULTS: Older adults had a disproportionate difficulty initiating movement when prevented from programming in advance. Older adults produced slower, less efficient movements, particularly when prevented from programming under greater precision requirements. CONCLUSIONS: The slower movements of older adults do not simply reflect a preference for online control, as older adults have less efficient movements when forced to reprogram their movements. Age-related motor slowing kinematically resembles that seen in patients with cerebellar dysfunction.

Antibodies to tumor necrosis factor alfa attenuate hepatic necrosis and inflammation caused by chronic exposure to ethanol in the rat.

Tumor necrosis factor (TNF)alpha, a pivotal cytokine involved in inflammation, is produced primarily by Kupffer cells in the liver. It has been shown that inactivation of Kupffer cells prevents alcohol-induced liver injury; therefore, the purpose of this study was to determine if neutralizing anti-TNF-alpha antibody is also effective. Male Wistar rats were exposed to ethanol (11 to 12 g x kg(-1) x d[-1]) continuously for up to 4 weeks via intragastric feeding using an enteral feeding model. Before ethanol exposure, polyclonal anti-mouse TNF-alpha rabbit serum was injected (2.0 mg/kg intravenously). There were no significant differences in body weight, mean ethanol concentration, or cyclic patterns of ethanol in urine when ethanol- and ethanol plus antibody-treated groups were compared. Expression of TNF-alpha and macrophage inflammatory protein 2 (MIP-2) messenger RNA (mRNA), determined using reverse transcription-polymerase chain reaction, was three- to four-fold higher in livers of ethanol-treated rats than in those of rats fed an ethanol-free, high-fat control diet. In addition, MIP-2 levels were also elevated when detected by Northern blot analysis. Anti-TNF-alpha antibody did not affect expression of mRNA for interleukin (IL) 1alpha, IL-6, transforming growth factor beta1, or TNF-alpha. However, MIP-2 mRNA expression, which is regulated by TNF-alpha, was decreased significantly by anti-TNF-alpha antibody treatment. Serum aspartate transaminase levels were elevated in ethanol-treated rats to 136 +/- 12 IU/L after 4 weeks but only reached 90 +/- 5 IU/L (P < .05) in rats treated with anti-TNF-alpha antibody. The hepatic inflammation and necrosis observed in ethanol-fed rats were attenuated significantly by antibody treatment, and steatosis was not. These results support the hypothesis that TNF-alpha plays an important role in inflammation and necrosis in alcohol-induced liver injury and that treatment with anti-TNF-alpha antibody may be therapeutically useful in this disease.

Modulation of NK cell cytolytic activity by macrophages in chronically exercise-stressed mice.

This study was designed to investigate the effects of moderate-intensity endurance training on basal natural killer (NK) cell cytolytic activity in murine splenocytes that were enriched for 1) NK1.1+ cells or 2) macrophages and NK1.1+ cells. Mice were assigned to sedentary (Sed), treadmill control (TM), or treadmill-trained (Trn) groups. Splenocyte number, the percentages of NK1.1+, large granular lymphocytes (NK1.1+, LGL-1+), and other subpopulations did not change in Trn mice. Approximately 70% of cells enriched for NK1.1+ expressed this surface antigen. Lytic units (LU) expressed per LGL-1+ cell were significantly lower in Trn [83.9 +/- 3.2 (SE)] compared with Sed (109.5 +/- 7.5) and TM (101.3 +/- 6.4) groups. When macrophages remained in the in vitro assay, LU per LGL-1(+) cell did not differ across groups. The results indicate that highly enriched NK1.1+ cells from Trn mice had lower NK cell activity compared with Sed mice. No differences in NK cell activity were observed when cells were enriched for NK1.1+ cells and macrophages. These findings support the hypothesis that macrophage modulation of NK cells may be one mechanism contributing to augmented basal NK cell activity in endurance-trained individuals.