Petia P. Simeonova

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.—Gal‐lucci, R. M., Simeonova, P. P., Matheson, J. M., Kommineni, C., Guriel, J. L., Sugawara, T., and Luster, M. I. Impaired cutaneous wound healing in interleukin‐6 – deficient and immunosuppressed mice. FASEB J. 14, 2525–2531 (2000)

Cardiovascular Effects of Pulmonary Exposure to Single-Wall Carbon Nanotubes

Background Engineered nanosized materials, such as single-wall carbon nanotubes (SWCNT), are emerging as technologically important in different industries. Objective The unique physical characteristics and the pulmonary toxicity of SWCNTs raised concerns that respiratory exposure to these materials may be associated with cardiovascular adverse effects. Methods In these studies we evaluated aortic mitochondrial alterations by oxidative stress assays, including quantitative polymerase chain reaction of mitochondrial (mt) DNA and plaque formation by morphometric analysis in mice exposed to SWCNTs. Results A single intrapharyngeal instillation of SWCNTs induced activation of heme oxygenase-1 (HO-1), a marker of oxidative insults, in lung, aorta, and heart tissue in HO-1 reporter transgenic mice. Furthermore, we found that C57BL/6 mice, exposed to SWCNT (10 and 40 μg/mouse), developed aortic mtDNA damage at 7, 28, and 60 days after exposure. mtDNA damage was accompanied by changes in aortic mitochondrial glutathione and protein carbonyl levels. Because these modifications have been related to cardiovascular diseases, we evaluated whether repeated exposure to SWCNTs (20 μg/mouse once every other week for 8 weeks) stimulates the progression of atherosclerosis in ApoE−/− transgenic mice. Although SWCNT exposure did not modify the lipid profiles of these mice, it resulted in accelerated plaque formation in ApoE−/− mice fed an atherogenic diet. Plaque areas in the aortas, measured by the en face method, and in the brachiocephalic arteries, measured histopathologically, were significantly increased in the SWCNT-treated mice. This response was accompanied by increased mtDNA damage but not inflammation. Conclusions Taken together, the findings are of sufficient significance to warrant further studies to evaluate the systemic effects of SWCNT under workplace or environmental exposure paradigms.

Physiological role of tumor necrosis factor α in traumatic muscle injury

The degenerative and regenerative roles of tumor necrosis factor α (TNF‐α), a pro‐inflammatory cytokine with pleiotropic functions, were investigated by using TNF receptor 1 and 2 double knockout (TNFR‐DKO) and TNF‐α antibody neutralized mice following traumatic freeze injury to the tibialis anterior muscle. In wild‐type control mice, TNF‐α mRNA transcripts and protein increased following injury and gradually returned to control (uninjured) levels by 13 days. A reduction in MyoD mRNA expression occurred in TNF–α‐deficient mice, although there were no visible differences in MyoD immunostaining or histological characteristics in regenerating muscles. At 5 days post‐injury, the reductions in isometric strength in TNFR‐DKO and TNF–α‐depleted mice did not differ from that of wild‐type mice but by 13 days after injury, the TNFR‐DKO and TNF–αdepleted mice exhibited strength deficits twice that of wild‐type mice (i.e., 27–31% vs 13%). Muscle injury was also accompanied by increased expression of interleukin‐6 (IL‐6), but IL–6‐deficient mice demonstrated MyoD expression and recovery of isometric strength similar to that of wild‐type mice. These data indicate that TNF‐α is involved in the recovery of muscle function after traumatic muscle injury, and this effect might be associated with modulation of muscle regulatory genes, including MyoD.

Work, Obesity, and Occupational Safety and Health

There is increasing evidence that obesity and overweight may be related, in part, to adverse work conditions. In particular, the risk of obesity may increase in high-demand, low-control work environments, and for those who work long hours. In addition, obesity may modify the risk for vibration-induced injury and certain occupational musculoskeletal disorders. We hypothesized that obesity may also be a co-risk factor for the development of occupational asthma and cardiovascular disease that and it may modify the workers response to occupational stress, immune response to chemical exposures, and risk of disease from occupational neurotoxins. We developed 5 conceptual models of the interrelationship of work, obesity, and occupational safety and health and highlighted the ethical, legal, and social issues related to fuller consideration of obesitys role in occupational health and safety.

Arsenic Enhancement of Skin Neoplasia by Chronic Stimulation of Growth Factors

Although numerous epidemiological studies have shown that inorganic arsenicals cause skin cancers and hyperkeratoses in humans, there are currently no established mechanisms for their action or animal models. Previous studies in our laboratory using primary human keratinocyte cultures demonstrated that micromolar concentrations of inorganic arsenite increased cell proliferation via the production of keratinocyte-derived growth factors. As recent reports demonstrate that overexpression of keratinocyte-derived growth factors, such as transforming growth factor (TGF)-alpha, promote the formation of skin tumors, we hypothesized that similar events may be responsible for those associated with arsenic skin diseases. Thus, the influence of arsenic in humans with arsenic skin disease and on mouse skin tumor development in transgenic mice was studied. After low-dose application of tetradecanoyl phorbol acetate (TPA), a marked increase in the number of skin papillomas occurred in Tg.AC mice, which carry the v-Ha-ras oncogene, that received arsenic in the drinking water as compared with control drinking water, whereas no papillomas developed in arsenic-treated transgenic mice that did not receive TPA or arsenic/TPA-treated wild-type FVB/N mice. Consistent with earlier in vitro findings, increases in granulocyte/macrophage colony-stimulating factor (GM-CSF) and TGF-alpha mRNA transcripts were found in the epidermis at clinically normal sites within 10 weeks after arsenic treatment. Immunohistochemical staining localized TGF-alpha overexpression to the hair follicles. Injection of neutralizing antibodies to GM-CSF after TPA application reduced the number of papillomas in Tg.AC mice. Analysis of gene expression in samples of skin lesions obtained from humans chronically exposed to arsenic via their drinking water also showed similar alterations in growth factor expression. Although confirmation will be required in nontransgenic mice, these results suggest that arsenic enhances development of skin neoplasias via the chronic stimulation of keratinocyte-derived growth factors and may be a rare example of a chemical carcinogen that acts as a co-promoter.

Cross-talk between lung and systemic circulation during carbon nanotube respiratory exposure. Potential biomarkers.

Nanotechnology is an emerging field that demands urgent development of adequate toxicology and risk assessment. The previous experimental data on carbon nanotube respiratory exposure strongly suggest the need for complex evaluation of potential toxicity. Our work demonstrates that after carbon nanotube deposition in the lung, acute local and systemic responses are activated and characterized by a blood gene and protein expression signature. The approach described here will foster the development of biomarkers for application in human screening of nanoparticle exposure.

Arsenic induces overexpression of growth factors in human keratinocytes.

Although epidemiological studies have shown that inorganic arsenicals are human skin carcinogens and induce hyperproliferation and hyperkeratosis, there is currently no known mechanism for their action or an established animal model for its study. We observed increased mRNA transcripts and secretion of keratinocyte growth factors, including granulocyte macrophage-colony stimulating factor (GM-CSF) and transforming growth factor-alpha (TGF alpha) and the proinflammatory cytokine tumor necrosis factor-alpha in primary human epidermal keratinocytes cultured in the presence of low micromolar concentrations of sodium arsenite. Treatment with sodium arsenite resulted in a significant increase in cell proliferation, as indicated by increases in cell numbers, c-myc gene expression, and incorporation of [3H]thymidine into cellular DNA. Studies of transcriptional regulation indicate that the rate of GM-CSF mRNA transcription is increased, while the elevated TGF alpha is likely the results of message stabilization. While a number of cytokine regulatory networks exist in the skin, studies utilizing neutralizing antibodies against the growth factors of interest indicate that inhibition of the arsenic-induced increase in TGF alpha results in a corresponding decrease in the gene expression and secretion of GM-CSF. The present studies demonstrate that growth-promoting cytokines and growth factors are induced in keratinocytes following treatment with arsenic and could play a significant role in arsenic-induced skin cancer.

Chemokine receptor CCR2 involvement in skeletal muscle regeneration

Chemokines, signaling through the CCR2 receptor, are highly expressed in injured skeletal muscle. Their target specificity depends on the cellular expression of the specific receptors. Here we demonstrate that, in freeze‐injured muscle, CCR2 co‐localized with Mac‐3, a marker of activated macrophages as well as with myogenin, a marker of activated muscle precursor cells. The degeneration/regeneration process in skeletal muscle of CCR2−/− and wild‐type mice was not significantly different at day 3. However in contrast to the regenerated muscle of the wild‐type mice, the muscle from CCR2−/− mice was characterized by impaired regeneration, inflammation, and fibrotic response at day 14, increased fat infiltration, fibrosis, and calcification at day 21, and impaired strength recovery until at least 28 days post‐injury. Consistently, the increased expression of Mac‐1 and TNF‐α was prolonged in the injured muscle of CCR2−/− mice. The expression pattern of the myogenic factors MyoD and myogenin was similar for both types of mice, while NCAM, which is associated with the initiation of fusion of muscle precursor cells, was more increased in the injured muscle of CCR2−/− mice. In conclusion, the study delineates that signaling through CCR2 is involved in muscle precursor cell activities necessary for complete and rapid regeneration of injured skeletal muscle.

Tumor Necrosis Factor α and Toxicology

The molecular cloning of a group of proteins, collectively referred to as cytokines, and including interleukins, chemokines, growth factors, colony stimulating factors, and tumor necrosis factors, has allowed for the increased understanding of the mechanisms for many disease processes as well as provided strategies for the development of novel therapies. Conceptually similar to hormones and peptides, this group of phylogenetically related molecules are also involved in various toxicological processes, including apoptosis, cell repair, and in particular inflammation. In this review, we offer a description of what many believe represents the primary regulatory cytokine, tumor necrosis factor (TNF)alpha and its role in toxicological processes. For over a decade it has been suspected that this molecule helps mediate the shock state induced by bacterial endotoxin and the wasting diathesis that typifies chronic diseases. Advances in molecular biology that have provided tools to modulate TNFalpha regulation and synthesis have allowed for the identification of additional roles for TNFalpha in homeostasis, cellular damage, and repair. This review provides a brief summary of our understanding of TNFalpha biology followed by a discussion of its role in toxicological responses. This is followed by specific examples of organ-specific and tissue-specific responses to chemical damage where TNFalpha has been implicated. The review concludes with a review of its implication in human risk assessment, particularly as it relates to genetic polymorphisms of TNFalpha expression and disease susceptibility.

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.