Electrochemical behaviour of anticancer drug lomustine and in situ evaluation of its interaction with DNA.

Electrochemical techniques were used to investigate the behavior of lomustine (CCNU) and its degradation in aqueous solution at a glassy carbon electrode (GCE). The in situ interaction of CCNU and chemically degraded CCNU (cdCCNU) with dsDNA was then investigated in dsDNA incubated solutions, using dsDNA electrochemical biosensors and comet assays. CCNU undergoes electrochemical reduction in two irreversible, diffusion-controlled, and pH-dependent redox processes, each with transfer of two electrons and one proton. At pH ≥ 10.1, the peak potential for the two processes was essentially pH-independent and involved only one electron. A mechanism was proposed for the reduction of CCNU in a neutral medium. In addition, it was found that CCNU underwent spontaneous degradation during incubation in aqueous solution, without the formation of electroactive degradation products. The degradation process was faster in basic media. Moreover, this pro-drug interacted with the DNA. Its metabolite(s) initially caused condensation of the double helix chains, followed by the unwinding of these chains. In addition, free guanine (Gua) was released from the dsDNA and oxidative damage to the DNA by the CCNU metabolite(s) was evidenced from the detection of 8-oxoGua and 2,8-oxoAde. These results were confirmed by the poly(dA)- and poly(dG)-polyhomonucleotide biosensors, which revealed the oxidative damage caused to both bases (guanine and adenine) of the dsDNA by the CCNU metabolite(s). The comet assay indicated breaks in the single strand DNA, complementing the results of the studies using differential pulse voltammetry. Conformational changes of dsDNA caused by CCNU and cdCCNU were confirmed using comet assays.

Neuropathic Pain Induced Alterations in the Opioidergic Modulation of a Descending Pain Facilitatory Area of the Brain.

Opioids play a major role at descending pain modulation but the effects of neuropathic pain on the brain opioidergic system remain understudied. Since descending facilitation is enhanced during neuropathic pain, we studied the opioidergic modulation of the dorsal reticular nucleus (DRt), a medullary pain facilitatory area, in the spared nerve injury (SNI) model of neuropathic pain. We first performed a series of behavioral experiments in naïve-animals to establish the role of µ-opioid receptor (MOR) in the effects of endogenous and exogenous opioids at the DRt. Specifically, we showed that lentiviral-mediated MOR-knockdown at the DRt increased sensitivity to thermal and mechanical stimuli while the MOR agonist DAMGO induced the opposite effects. Additionally, we showed that MOR-knockdown and the pharmacological blockade of MOR by CTAP at the DRt decreased and inhibited, respectively, the analgesic effects of systemic morphine. Then, we performed in vivo microdialysis to measure enkephalin peptides in the DRt and evaluated MOR expression in the DRt at mRNA, protein and phosphorylated form levels by quantitative real-time PCR and immunohistochemistry, respectively. SNI-animals, compared to sham control, showed higher levels of enkephalin peptides, lower MOR-labeled cells without alterations in MOR mRNA levels, and higher phosphorylated MOR-labeled cells. Finally, we performed behavioral studies in SNI animals to determine the potency of systemic morphine and the effects of the pharmacologic and genetic manipulation of MOR at the DRt. We showed a reduced potency of the antiallodynic effects of systemic morphine in SNI-animals compared to the antinociceptive effects in sham animals. Increasing MOR-cells at the DRt of SNI-animals by lentiviral-mediated MOR-overexpression produced no effects on mechanical allodynia. DAMGO induced anti-allodynia only after MOR-overexpression. These results show that MOR inhibits DRt pain facilitatory actions and that this action contributes to the analgesic effects of systemic opioids. We further show that the inhibitory function of MOR is impaired during neuropathic pain. This is likely due to desensitization and degradation of MOR which are adaptations of the receptor that can be triggered by MOR phosphorylation. Skipping counter-regulatory pathways involved in MOR adaptations might restore the opioidergic inhibition at pain facilitatory areas.

Increased noradrenergic neurotransmission to a pain facilitatory area of the brain is implicated in facilitation of chronic pain.

BACKGROUND: Noradrenaline reuptake inhibitors are known to produce analgesia through a spinal action but they also act in the brain. However, the action of noradrenaline on supraspinal pain control regions is understudied. The authors addressed the noradrenergic modulation of the dorsal reticular nucleus (DRt), a medullary pronociceptive area, in the spared nerve injury (SNI) model of neuropathic pain. METHODS: The expression of the phosphorylated cAMP response element-binding protein (pCREB), a marker of neuronal activation, was evaluated in the locus coeruleus and A5 noradrenergic neurons (n = 6 rats/group). pCREB was studied in noradrenergic DRt-projecting neurons retrogradely labeled in SNI animals (n = 3). In vivo microdialysis was used to measure noradrenaline release in the DRt on nociceptive stimulation or after DRt infusion of clonidine (n = 5 to 6 per group). Pharmacology, immunohistochemistry, and western blot were used to study α-adrenoreceptors in the DRt (n = 4 to 6 per group). RESULTS: pCREB expression significantly increased in the locus coeruleus and A5 of SNI animals, and most noradrenergic DRt-projecting neurons expressed pCREB. In SNI animals, noradrenaline levels significantly increased on pinprick (mean ± SD, 126 ± 14%; P = 0.025 vs. baseline) and acetone stimulation (mean ± SD, 151 ± 12%; P < 0.001 vs. baseline), and clonidine infusion showed decreased α2-mediated inhibitory function. α1-adrenoreceptor blockade decreased nociceptive behavioral responses in SNI animals. α2-adrenoreceptor expression was not altered. CONCLUSIONS: Chronic pain induces brainstem noradrenergic activation that enhances descending facilitation from the DRt. This suggests that antidepressants inhibiting noradrenaline reuptake may enhance pain facilitation from the brain, counteracting their analgesic effects at the spinal cord.

GABA acting on GABAB receptors located in a medullary pain facilitatory area enhances nociceptive behaviors evoked by intraplantar formalin injection.

The dorsal reticular nucleus (DRt) plays a key role in facilitation of nociceptive transmission at the spinal cord. In this study, we evaluated the mechanisms involved in GABA-mediated control of the DRt focusing on the role of local GABAB receptors. First, we used in vivo microdialysis to study the release of GABA in the DRt during the course of the formalin test. An increase of GABA levels in comparison with baseline values was detected in the second phase of the test. Because we previously showed that GABAB receptors are expressed by opioidergic DRt neurons, which respond to nociceptive stimuli and inhibit spinally projecting DRt neurons involved in descending pronociception, we then interfered with local GABAB receptors using gene transfer and pharmacological approaches. Lentiviral-mediated knockdown of GABAB1a expression decreased nociceptive responses during the second phase of the test. Local administration of the GABAB receptor antagonist CGP 35348 also decreased nociceptive responses in the second phase of the test, whereas the opposite was detected after injection of the GABAB agonist baclofen. Finally, we determined the GABAergic afferents of the DRt, namely those arising from its main brain afferents, which are located at the telencephalon and diencephalon. For that purpose, we combined retrograde tract-tracing from the DRt with immunodetection of glutamate decarboxylase, the GABA-synthesizing enzyme. The higher numbers of retrogradely labelled glutamate decarboxylase-immunoreactive neurons were located at insular, somatosensory, and motor cortices. Collectively, the results suggest that GABA acting on GABAB receptors may enhance pain facilitation from the DRt during inflammatory pain.