Patrizia Bonfanti

Non-Neuronal Cell Modulation Relieves Neuropathic Pain: Efficacy of the Endogenous Lipid Palmitoylethanolamide

We have previously shown that the endogenous lipid palmitoylethanolamide (PEA) induced relief of neuropathic pain through an action upon receptors located on the nociceptive pathway. Recently, it has been proposed that immune cells, in particular mast cells, and microglia, by releasing algogen mediators interact with neurons to alter pain sensitivity thereby contributing to the development and maintenance of chronic pain states. The aim of this work was to explore whether the anti-nociceptive properties of PEA might be accompanied by modulation of these non-neuronal cells. Mice were subjected to a chronic constriction injury model of neuropathic pain and treated with PEA. The data show that at the earlier (3 days) time-point after nerve injury there was a substantial recruitment of mast cells whose activation was not yet pronounced. In contrast, at the later time point (8 days) there was no further increase in mast cell number, but rather a marked activation of these cells. An up-regulation of activated microglia was found in the spinal cord of neuropathic pain mice. PEA delayed mast cell recruitment, protected mast cells against degranulation and abolished the nerve growth factor increase in sciatic nerve concomitantly preserving the nerve from degeneration, while reducing microglia activation in the spinal cord. These findings support the idea that non-neuronal cells may be a valuable pharmacological target to treat neuropathic pain since the current neuronal-direct drugs are still unsatisfactory. In this context PEA could represent an innovative molecule, combining a dual analgesic activity, both on neurons and on nonneuronal cells.

The use of in vitro fertilization in the Frog Embryo Teratogenesis Assay in Xenopus (FETAX) and its applications to ecotoxicology

The Frog Embryo Teratogenesis Assay in Xenopus (FETAX) is a powerful assay for the presence of developmental toxicants in the environment that uses Xenopus embryos. We have applied the test to evaluate a water purification system by testing and comparing the input and the output waters.

Chronic treatment with polychlorinated biphenyls (PCB) during pregnancy and lactation in the rat: Part 1: Effects on somatic growth, growth hormone-axis activity and bone mass in the offspring.

Polychlorinated biphenyls (PCBs) are pollutants detected in animal tissues and breast milk. The experiments described in the present paper were aimed at evaluating whether the four PCB congeners most abundant in animal tissues (PCB-138, -153, -180 and -126), administered since fetal life till weaning, can induce long-term alterations of GH-axis activity and bone mass in the adult rat. We measured PCB accumulation in rat brain and liver, somatic growth, pituitary GH expression and plasma hormone concentrations at different ages. Finally, we studied hypothalamic somatostatin expression and bone structure in adulthood, following long-term PCB exposure. Dams were treated during pregnancy from GD15 to GD19 and during breast-feeding. A constant reduction of the growth rate in both male and female offspring from weaning to adulthood was observed in exposed animals. Long-lasting alterations on hypothalamic-pituitary GH axis were indeed observed in PCB-exposed rats in adulthood: increased somatostatin expression in hypothalamic periventricular nucleus (both males and females) and lateral arcuate nucleus (males, only) and decreased GH mRNA levels in the pituitary of male rats. Plasma IGF-1 levels were higher in PCB-exposed male and female animals as compared with controls at weaning and tended to be higher at PN60. Plasma testosterone and thyroid hormone concentrations were not significantly affected by exposure to PCBs. In adulthood, PCBs caused a significant reduction of bone mineral content and cortical bone thickness of tibiae in male rat joint to increased width of the epiphyseal cartilage disk. In conclusion, the developmental exposure to the four selected PCB compounds used in the present study induced far-reaching effects in the adult offspring, the male rats appearing more sensitive than females.

In vitro biological systems as models to evaluate the toxicity of pesticides

Abstract Recently the use of in vitro systems has gained acceptance for toxicological research, since they offer several advantages. We have used primary and immortalized cell cultures to evaluate the effect of the fungicide Methyl 1-(butylcarbamoyl)benzimidazole-2-carbamate (Benomyl), the insecticide 0–2-diethylamino-6-methylpyrimidine-4-yl—0,0-dimethyl phosphorothioate (Pirimiphos-methyl), the herbicide 4-chloro-2-methylphenoxyacetic acid (MCPA). Benomyl resulted responsible for microtubular disorganization in time-dose dependent manner, and for glutathione depletion. Pirimiphos-methyl, alone or combined with Benomyl. had no effect on microtubule organization, but reinforced glutathione depletion. MCPA exerted its primary effect on mitochondria, increasing the mitochondrial membrane potential and inhibiting the ATP-synthesizing ATPase. Flow cytometric analysis suggests that MCPA affects C3H cell cycle and induces the formation of type II foci in standard transformation assay. The results presented demon...

Molecular approaches to evaluate pollutants

Many organisms are in use to test pollutants and their extensive variability clearly emerges from reviews since researchers in the world are involved in continuous effort to set up new assays and to improve those already in use. In the present paper we focus the attention on the mixed function oxidase system and the DNA adduct formation which are two biomarkers widely used and extensively studied in mammals and fish by different Authors. We compare their results with the ones we obtained in amphibians, which result to be a good model. Moreover we present some significative results obtained by the use of cultured cell lines to test the herbicide MCPA. The results obtained demonstrate that the amphibian Xenopus is a suitable indicator for induction of cytochrome P-450 by B[a]P as well as for production of DNA adducts. Cultured cells evidenced that cytoskeletal array and thiol proteins are molecular targets of the herbicide used, demonstrating that risk assessment can be properly analysed in in vitro systems.

Induction of cytochrome P4501A isoform in Xenopus laevis is a valid tool for monitoring the exposure to benzo[a]pyrene

In mammals polycyclic aromatic hydrocarbons (PAHs) are metabolized by the enzymes of the cytochrome P-4501A family expressed in specific cell types. Recently the expression of both CYP1A1 and CYP1A2 transcripts has been demonstrated in liver of 3-MC treated rainbow trout. In the present study induction of CYP1A was investigated by immunoblot analysis in adults and embryos of Xenopus laevis treated with benzo[a]pyrene (B[a]P). The data obtained showed that polyclonal antibodies, raised against rat cytochrome P-4501A1, recognized a single band with apparent molecular weight between 55 kDa and 66 kDa as rat CYP1A both in adult liver microsomes and embryo homogenates. The assessment of the metabolic activity, performed with ethyl acetate extraction of treated embryo homogenate demonstrated that X. laevis early developmental stages (stage 35 and stage 48) are competent to convert B[a]P into its metabolites. These results suggest that X. laevis is a sensitive model to evaluate fresh water pollution.

Do Nanoparticle Physico-Chemical Properties and Developmental Exposure Window Influence Nano ZnO Embryotoxicity in Xenopus laevis?

The growing global production of zinc oxide nanoparticles (ZnONPs) suggests a realistic increase in the environmental exposure to such a nanomaterial, making the knowledge of its biological reactivity and its safe-by-design synthesis mandatory. In this study, the embryotoxicity of ZnONPs (1–100 mg/L) specifically synthesized for industrial purposes with different sizes, shapes (round, rod) and surface coatings (PEG, PVP) was tested using the frog embryo teratogenesis assay-Xenopus (FETAX) to identify potential target tissues and the most sensitive developmental stages. The ZnONPs did not cause embryolethality, but induced a high incidence of malformations, in particular misfolded gut and abdominal edema. Smaller, round NPs were more effective than the bigger, rod ones, and PEGylation determined a reduction in embryotoxicity. Ingestion appeared to be the most relevant exposure route. Only the embryos exposed from the stomodeum opening showed anatomical and histological lesions to the intestine, mainly referable to a swelling of paracellular spaces among enterocytes. In conclusion, ZnONPs differing in shape and surface coating displayed similar toxicity in X. laevis embryos and shared the same target organ. Nevertheless, we cannot exclude that the physico-chemical characteristics may influence the severity of such effects. Further research efforts are mandatory to ensure the synthesis of safer nano-ZnO-containing products.

Responsiveness of hepatic and cerebral cytochrome P450 in rat offspring prenatally and lactationally exposed to a reconstituted PCB mixture

Perinatal polychlorinated biphenyl (PCB) exposures still remain a serious health concern because offspring receive PCB burden from mother during vulnerable processes of development. Since cytochrome P450 (CYP) represents a toxicological endpoint, in the present study, representing an extended investigation of a previous multitasked one, we explored the long‐term responsiveness of CYP1A and CYP2B isoforms by Western blot analysis in liver and whole brain of lactating (PN12), weaning (PN21), and adult offspring (PN60) rats prenatally and lactationally exposed to a reconstituted PCB mixture (RM) of noncoplanar PCB138, 153, 180, and coplanar PCB126 congeners. We chose highly chlorinated PCBs instead of lower chlorinated one, because their recalcitrance to biotransformation makes easy their accumulation/persistence in tissues and breast milk. Dioxin‐like congener PCB126 binding aryl hydrocarbon receptor (AHR) is responsible of many toxic effects. Pregnant Sprague–Dawley dams with high affinity AHR received subcutaneous injection of RM (10 mg/kg body weight) daily during gestation (days 15–19) and twice a week during breast‐feeding. The results evidenced a transfer of PCBs to neonates through milk and a significant responsiveness of hepatic CYP in both mothers and offspring. In liver of exposed progeny, CYP isoforms exhibited a significant increment at PN12 (70% over control) and at PN21 (270% over control). Contrary to dams, in adult PCB offspring CYP levels showed a decline up to values similar to those of control. This transient developmental responsiveness of CYP isoforms in offspring liver reflects roughly the time course of hepatic PCB levels previously reported. Even if congeners were detected in brain, we failed in evidencing a responsiveness of CYP isoforms probably because of region‐specific CYP expression in this organ. In conclusion, induction of offspring hepatic CYP is index of liver PCB burden, and despite the insensitivity of whole brain CYP we cannot exclude brain vulnerability toward PCB.

Brush border and cell adhesion of insect enterocytes have basic similarities with those of vertebrates

The molecular architecture of the brush border (BB) cytoskeleton of the vertebrate intestinal cells results to be well defined in its components. From the comparison of the protein profiles of chicken and larval BB extracts of Manduca sexta (Insecta, Lepidoptera) it emerges that cytoskeletal major components are the same and they have a similar localization in immunolabeled tissues. The smooth septate junctions (SSJs) of the midgut cells are localized in the most apical regions, where vertebrate intestinal cells exhibit the zonula occludens. We demonstrate that α-actinin, vinculin and myosin II are involved in the actin filaments-SSJ interaction. The insertion of microfilament bundles into submembranous F-actin/α-actinin/ vinculin complexes, discovered and described almost exclusively in vertebrate junctions of adherens type, results to be a more general system including the insect SSJs, that we have analyzed.