Alessandra Bruccoleri

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.

Cellular localization and temporal elevation of tumor necrosis factor-α, interleukin-1α, and transforming growth factor-β1 mRNA in hippocampal injury response induced by trimethyltin

Abstract: In certain pathologic states, cytokine production may become spatially and temporally dysregulated, leading to their inappropriate production and potentially detrimental consequences. Tumor necrosis factor‐α (TNF‐α), interleukin (IL)‐1, IL‐6, and transforming growth factor‐β (TGF‐β) mediate a range of host responses affecting multiple cell types. To study the role of cytokines in the early stages of brain injury, we examined alterations in the 17‐day‐old mouse hippocampus during trimethyltin‐induced neurodegeneration characterized by neuronal necrosis, microglia activation in the dentate, and astrocyte reactivity throughout the hippocampus. By 24 h after dosing, elevations in mRNA levels for TNF‐α, IL‐1α, IL‐1β, and IL‐6 mRNA were seen. TGF‐β1 mRNA was elevated at 72 h. In situ hybridization showed that TNF‐α and IL‐1α were localized to the microglia, whereas TGF‐β1 was expressed predominantly in hippocampal pyramidal cells. Intercellular adhesion molecule‐1, EB‐22, Mac‐1, and glial fibrillary acidic protein mRNA levels were elevated within the first 3 days of exposure in the absence of increased inducible nitric oxide synthetase and interferon‐γ mRNA. These data suggest that pro‐inflammatory cytokines contribute to the progression and pattern of neuronal degeneration in the hippocampus.

Chemical-induced hippocampal neurodegeneration and elevations in TNF?, TNF?, IL-1?, IP-10, and MCP-1 mRNA in osteopetrotic (op/op) mice

The osteopetrotic (op/op) mouse, deficient in biologically active colony stimulating factor 1 (CSF‐1), was used to examine the role of microglia in chemical‐induced trauma. Op/op mice and normal phenotype littermates (non‐op/op) received an acute i.p. injection of the hippocampal toxicant, trimethyltin hydroxide (TMT; 1.5 or 2.0 mg/kg). At 2.0 mg/kg, both mice displayed severe degeneration of dentate granule neurons. At 1.5 mg/kg, non‐op/op mice showed a limited punctate pattern of neuronal death while op/op mice showed prominent neuronal death. TMT‐induced astrocyte reactivity was similar in both groups. RNase protection assays were conducted on hippocampal tissue at 24 hr post‐TMT. Elevations were seen in mRNA levels for the host response genes: intercellular cell adhesion molecule (ICAM‐1; non‐op/op 80%, op/op 85%), the protease inhibitor EB22 (non‐op/op 60%, op/op 300%), and glial fibrillary acidic protein (GFAP; non‐op/op 300%, op/op 480%) within 24 hr. Macrophage‐1 antigen (Mac‐1) mRNA levels were lower in all op/op mice and were not induced by TMT exposure. Macrophage inflammatory protein (MIP)‐1α and MIP‐1β mRNA levels were elevated in non‐op/op mice while mRNA levels for interferon inducible protein (IP‐10) and monocyte chemoattractant protein (MCP‐1) were elevated in op/op mice. Tumor necrosis factor alpha (TNFα) mRNA levels were significantly elevated in both non‐op/op (100%) and op/op (600%) mice. TNFβ mRNA levels in op/op mice were elevated 200% and interleukin 1alpha (IL‐1α) 150%. Reverse transcriptase polymerase chain reaction (RT‐PCR) showed a TMT‐induced elevation in INFα and INFβ mRNA levels and no elevation of INFγ. mRNA levels of the CSF‐1 receptor, c‐fms, were unaltered. J. Neurosci. Res. 62:146–155, 2000. Published 2000 Wiley‐Liss, Inc.

Effect of dexamethasone on elevated cytokine mRNA levels in chemical-induced hippocampal injury.

An acute administration of the hippocampal toxicant trimethyltin (TMT) produced a specific pattern of neuronal necrosis in dentate granule cells with accompanying astrogliosis and initiation of a cytokine response within 24 hours. The purpose of this study was to examine the effects of the anti‐inflammatory agent, dexamethasone (DEX), on the pattern of cytokine expression and neuronal degeneration occurring after an acute TMT injection. Dexamethasone (0.2 mg/kg or 10 mg/kg) was administered to 21‐day‐old male mice 1 hour prior to an injection of TMT hydroxide (2.5 mg/kg, i.p.). Mice receiving 0.2 mg/kg DEX received a second injection 6 hours after TMT. Twenty‐four hours later, neuronal necrosis and astrogliosis were assessed and found to be similar in animals treated with TMT, either in the presence or absence of dexamethasone. Pretreatment with dexamethasone failed to prevent the neurodegeneration and astrogliosis. The TMT‐induced injury response was represented in elevations of mRNA levels for the injury‐associated host response genes glial fibrillary acidic protein (GFAP), EB22/5.3, and intercellular adhesion molecule‐1 (ICAM‐1). The combination of DEX and TMT produced increased elevation in mRNA levels for EB22/5.3 and ICAM, while GFAP levels remained the same as with TMT alone. The injury response from TMT was accompanied by elevations in mRNA levels for the cytokines tumor necrosis factor (TNF) α, TNFβ, and interleukin (IL)‐1α. Treatment with dexamethasone prior to TMT resulted in significantly elevated levels of TNFα, TNFβ, and IL‐1α as compared to TMT alone. These data represent the inability of glucocorticoids to downregulate the injury response in rat hippocampus following a systemic injection of TMT and suggest a stimulation and “priming” of hippocampal cells by dexamethasone. J. Neurosci. Res. 57:916–926, 1999.

Role of keratinocyte-derived cytokines in chemical toxicity.

Following appropriate stimulation, such as with tumor promoters, ultraviolet light or various chemical agents, keratinocytes synthesize and secrete cytokines which can mediate or participate in dermatotoxic responses such as inflammation, hyperkeratosis, hypersensitivity and skin cancer. We have determined the qualitative and quantitative cytokine response in primary human keratinocyte cultures following exposure to several non-sensitizing contact irritants, sensitizers and ulcerative agents as well as a skin carcinogen. The chemicals were also administered to mice to assess whether the dermatotoxic response correlated with the in vitro production of keratinocyte-derived cytokines. Due to the complex cellular interactions that occur in the skin, it was not possible to identify specific cytokine profiles for most of the classes of dermatotoxic agents studied. However, the non-sensitizing contact irritants produced relative increases in the synthesis and secretion of the proinflammatory cytokines, interleukin-1 and tumor necrosis factor-alpha, as well as the neutrophil chemotactic cytokine, interleukin-8 compared to the other chemical agents. While ulcerative compounds as well as irritants elicited neutrophils to the site of chemical application when applied to the mouse skin, time-dependent and chemical-specific patterns of inflammation were detected. Treatment of human keratinocyte cultures with arsenic, a human skin carcinogen, resulted in a unique cytokine profile characterized by induction of growth factors, including transforming growth factor-alpha and granulocyte-macrophage colony stimulating factor. Treatment of v-Ha-ras transgenic mice, an animal model for skin cancer, with arsenic caused an increase in the number of papillomas as well as overexpression of these growth factors suggesting that they participate in arsenic-induced skin papilloma development. These studies indicate a diverse role exists for keratinocyte-derived cytokines in dermatotoxic actions.

Differential modulation of hippocampal chemical-induced injury response by ebselen, pentoxifylline, and TNFα-, IL-1α-, and IL-6-neutralizing antibodies

The proinflammatory cytokines tumor necrosis factor (TNFα), interleukin‐1 (IL‐1α), and interleukin‐6 (IL‐6) have been associated with various models of hippocampal damage. To examine their role in initiation of an acute hippocampal injury response, 21‐day‐old male CD‐1 mice received an acute intraperitoneal (i.p.) injection of trimethyltin hydroxide (TMT; 2.0 mg/kg) to produce necrosis of dentate granule neurons, astrocyte, and microglia reactivity. Tremors and intermittent seizures were evident at 24 hr. Intercellular adhesion molecule‐1 (ICAM‐1), glial fibrillary acidic protein (GFAP), anti‐apoptotic TNFα‐inducible early response gene (A‐20), macrophage inflammatory protein (MIP)‐1α, TNFα, IL‐1α, IL‐6, and caspase 3 mRNA levels were significantly elevated. Pretreatment with the antioxidant, ebselen, decreased ICAM‐1, A‐20, and TNFβ elevations. Pentoxifylline blocked elevations in A‐20 and decreased elevations in GFAP mRNA levels. Neither prevented histopathology or behavioral effects. Intracisternal injection of TNFα‐neutralizing antibody significantly inhibited both behavioral effects and histopathology. RNase protection assays showed that TMT‐induced elevations in mRNA levels for ICAM‐1, A‐20, GFAP, MIP‐1α, IL‐1α, TNFα, TNFβ, and caspase 3 were blocked by anti‐TNFα. These data demonstrate a significant role for TNFα in an acute neuro‐injury in the absence of contribution from infiltrating cells. The cerebellum shows limited if any damage after TMT; however, in combination with the i.c.v. injection, elevations were seen in GFAP and in EB‐22, a murine acute‐phase response gene homologous to the alpha (1)‐antichymotrypsin gene. Elevations were similar for artificial cerebral spinal fluid and anti‐IL‐1α, and significantly increased with anti‐TNFα, anti‐IL‐6, or the combination of antibodies. Responses seen in the cerebellum suggest synergistic interactions between the baseline state of the cell and manipulations in the cytokine environment. Data suggests a role for TNFα in the pathogenesis of hippocampal injury induced by TMT. Published 2003 Wiley‐Liss, Inc.

Increase in brain stem cytokine mRNA levels as an early response to chemical-induced myelin edema

This study examined the early response of pro-inflammatory and regulatory cytokines in the mouse brain following triethyltin (TET)-induced myelin injury characterized by edematous vacuolation. Following an acute intraperitoneal injection of triethyltin (TET) sulfate (3 mg/kg) to 17-day old CD1 mice, significant increases in brain stem TNF-alpha and IL-1alpha mRNA levels occurred at 6 and 24 h, respectively with elevations in TGF-beta1 and MIP-1alpha at 1 h. In the cortex, responses were limited to elevations at 6 h in TNF-alpha, TGF-beta1 and MIP-1alpha. These data suggest that a chemokine/cytokine response can occur with minimal alterations to the integrity of the myelin sheath and may contribute to the initial signaling mechanisms associated with demyelinating disorders.