Intrathecal administration of botulinum toxin type A improves urinary bladder function and reduces pain in rats with cystitis.

BACKGROUND: Botulinum toxin A (Onabot/A) has been shown to have an antinociceptive effect. This might be due to an impairment of sensory nerves not only in the peripheral but also in the central nervous system. In this work, we analysed both systems by studying the effect of intrathecal (i.t.) administration of botulinum toxin A in an animal model of bladder pain and hyperactivity induced by cyclophosphamide (CYP). METHODS: Rats were implanted with an i.t. catheter at the L6 segment. Bladder pain was induced by intraperitoneal (i.p.) injection of CYP. Five experimental groups were created: (1) Saline i.p. + i.t.; (2) Onabot/A i.t.; (3) CYP i.p. + saline i.t.; (4) CYP i.p. + Onabot/A i.t. 48 h after CYP; and (5) Onabot/A i.t. 30 days. Mechanical sensitivity was assessed in the abdomen and hindpaws. Motor activity was observed in an open-field arena. Bladder reflex activity was evaluated by cystometry. At the end, bladders and spinal cord were immunoreacted (IR) against cleaved SNAP-25 (cSNAP-25), c-Fos, p-ERK, calcitonin gene-related peptide (CGRP) and GAP43. RESULTS: The toxin reduced pain symptoms, bladder hyperactivity, expression of neuronal activation markers and CGRP, typically up-regulated in this inflammatory model. The presence of cSNAP-25 was detected in the spinal cord and bladder fibres from animals treated with Onabot/A. No somatic or visceral motor impairments were observed. CONCLUSIONS: Our findings suggest that i.t. Onabot/A has a strong analgesic effect in a model of severe bladder pain. This route of administration can be further explored to treat intractable forms of pain.

TRPA1 mediates bladder hyperalgesia in a mouse model of cystitis.

Urinary bladder pain is a primary symptom associated with interstitial cystitis/painful bladder syndrome. We used systemic injections of cyclophosphamide (CYP), an alkylating antineoplastic agent, to induce cystitis and examine the roles of 2 channels previously demonstrated to be required for inflammatory visceral hyperalgesia: transient receptor potential vanilloid-1 (TRPV1) and ankyrin-1 (TRPA1). Injection of CYP (100 mg/kg, i.p.) every other day for 5 days was accompanied by bladder edema and urothelial ulceration, but without significant plasma extravasation or infiltration of neutrophils. Toluidine blue staining showed a significant increase in the number of degranulated bladder mast cells after CYP treatment. Despite this mild pathology, CYP-treated mice exhibited bladder hyperalgesia 1 day after the final injection that persisted 7 days later. Although many previous studies of visceral hyperalgesia have reported changes in dorsal root ganglion neuron TRPV1 expression and/or function, we found no change in bladder afferent TRPV1 expression or sensitivity on the basis of the percentage of bladder afferents responsive to capsaicin, including at submaximal concentrations. In contrast, the percentage of bladder afferents expressing functional TRPA1 protein (i.e., those responsive to mustard oil) increased ∼2.5-fold 1 day after CYP treatment, and remained significantly elevated 7 days later. Moreover, bladder hyperalgesia was reversed by acute treatment with the TRPA1 antagonist HC-030031 (300 mg/kg, i.p.). Our results indicate that CYP-induced bladder hyperalgesia can be induced without robust inflammation or changes in primary afferent TRPV1. However, significant changes were observed in TRPA1 expression, and blockade of TRPA1 alleviated CYP-induced bladder hyperalgesia.

Respuesta de ratones Balb/c frente a ciclofosfamida y bleomicina en el ensayo cometa alcalino de linfocitos de sangre periférica / Balb/c mice response against cyclophosphamide and bleomycin in the alkaline comet assay of peripheral blood lymphocytes

En este artículo se evaluó el efecto mutágenico de la ciclofosfamida y bleomicina, con el objetivo de armonizar el número de exposiciones al ser utilizadas como controles positivos en ensayos in vivo de genotoxicidad, mediante el ensayo cometa alcalino. Se realizó en linfocitos de sangre periférica, utilizando 10 ratones/grupo/sexo de la línea Balb/c como biomodelo experimental. Fueron formados 5 grupos experimentales/sexo, el primero administrado con NaCl al 0,9% por vía intraperitoneal (i.p) como control negativo. El segundo y el tercero administrados con ciclofosfamida por vía i.p, con diseños de tratamientos diferentes en dosis de 50 mg/kg. El cuarto y quinto grupo fueron administrados con bleomicina por vía i.p, igualmente en dos diseños de tratamientos diferentes en dosis de 20 mg/kg. El mayor valor de inducción de daño se obtuvo con el uso de la ciclofosfamida y bleomicina, ambas en el diseño de administración de 48 y 24 horas antes de la eutanasia. Este estudio será aplicable a la evaluación de drogas que no han sido exploradas en el ámbito de la antigenotoxicidad y genotoxicidad in vivo. Además permitirá contar con un mayor conocimiento acerca de este ensayo, favoreciendo su validación (AU)

In this article were evaluated the mutagenic effect of cyclophosphamide and bleomycin, with the objective of harmonizing the number of exhibitions when being used as positive controls on in vivo genotoxicity assay, by means of alkaline comet assay. It was carried out in peripheral blood lymphocytes, using 10 mice/group/sex of the Balb/c line as experimental biomodel. We were formed 5 experimental groups per sex. The first group was administered with NaCl 0,9 % by intraperitoneal (i.p) route. The second and third groups were administered with cyclophosphamide by i.p route, with designs of different treatments at doses of 50 mg/kg. The fourth and fifth groups were administered with bleomycin by i.p route, equally in two designs of different treatments at doses of 20 mg/kg. The bigger inductions of damage were obtained with the use of the cyclophosphamide and bleomycin, both in the design of 48 and 24 hours administration before the euthanasia. This study will be applicable to the drugs evaluation that they have not been explored in to the in vivo antigenotoxicity and genotoxicity environment. It will also allow having a bigger knowledge about this assay, favoring their validation (AU)

Reducing workplace cytotoxic surface contamination using a closed-system drug transfer device.

BACKGROUND: The potential for staff exposure to antineoplastic agents exists in the workplace despite current recommended safe handling procedures. Reliance on cytotoxic drug safety cabinets (CDSC) to provide total protection from exposure to hazardous drugs is insufficient. Preventing workplace contamination is the best strategy to minimise exposure. PhaSeal is a commercially available system for ensuring the leak-free transfer of hazardous drugs, fitting both the NIOSH and ISOPP definitions of a closed system. To date, there have been no published studies examining the use of a closed system drug transfer device (PhaSeal) under Australian conditions.The purpose of this study is to determine the impact of a closed system drug transfer device on cytotoxic surface contamination in the cytotoxic preparation areas of two Australian metropolitan public hospitals. METHOD: This was a pre- and post-intervention study in which chemical contamination was tested at baseline then at five and 12 months after the introduction of the a closed system drug transfer device. Cyclophosphamide was used as a surrogate marker for all cytotoxic drugs. Surface wipe sampling was performed at specified sites within the cytotoxic suite using a standardized technique. Commercial products of cyclophosphamide were also sampled. RESULTS: After five months, contamination was reduced in 13 of the 22 sites sampled (59%), with four of these samples showing undetectable levels of contamination. Two other site samples (9%) remained unchanged. The total contamination of surfaces tested was reduced by 24%. After five months hospital 1 withdrew from the study. After 12 months, surface contamination was reduced in 75% of sample sites. The total contamination of surfaces tested was reduced by 68%. The wipes of the external surface of commercial products detected cyclophosphamide contamination. CONCLUSION: When used inside a CDSC, the closed system drug transfer device PhaSeal further reduces surface contamination, in some instances to undetectable levels.

Long-term clinical outcome after accidental overdose of multiple chemotherapeutic agents.

Treatment of non-Hodgkin's lymphoma with the CHOP regimen consists of intravenous cyclophosphamide 750 mg/m2 (day 1), intravenous doxorubicin 50 mg/m2 (day 1), intravenous vincristine 1.4 mg/m2 (day 1), and oral prednisone 100 mg (days 1-5). This regimen is administered in cycles of approximately 3 weeks; a total course of treatment consists of six cycles. We report the case of a 23-year-old woman with diffuse large-cell lymphoma who received an accidental overdose of this chemotherapeutic regimen. The first cycle of her CHOP regimen was initiated (day 1) in our outpatient unit; she was then discharged home. Unfortunately, the patient went to another hospital located in the small city where she lived, and all remaining doses of the total course of treatment were administered over the next 5 consecutive days, with no interruption in therapy. She had received cumulative doses of cyclophosphamide 6000 mg, doxorubicin 420 mg, and vincristine 12 mg. She was transferred to our hospital after she developed pancytopenia, fever, and ileus. With the help of intensive supportive care and symptomatic treatment, the patient recovered and was discharged home after a hospital stay of 25 days. After 56 months, she was free of disease and treatment-related toxicities. Only experienced clinicians should administer chemotherapy, and thorough records must be kept to document the chemotherapy administered, dosages, dates of administration, the procedure used, and the schedule of cycles administered.

A mouse-based strategy for cyclophosphamide pharmacogenomic discovery.

Genome-wide mapping approaches are needed to more fully understand the genetic basis of chemotherapy response. Because of technical and ethical limitations, cancer pharmacogenomics has not yet benefited from traditional robust familial genetic strategies. We have therefore explored the use of the inbred mouse as a genetic model system in which to study response to the cytotoxic agent cyclophosphamide. Multiple phenotypes have been assessed in response to cyclophosphamide in up to 19 inbred mouse strains, including in vitro hematopoietic progenitor cell toxicity and the mobilization of hematopoietic progenitor cells into peripheral blood. Hematopoietic progenitor cell toxicity in vitro varied 2-fold among strains, whereas in vivo progenitor cell mobilization varied almost 75-fold among strains. Males mobilized more hematopoietic progenitor cells than did females, and the low-mobilization phenotype was dominant to the high-mobilization phenotype in F1 hybrid animals. In an initial attempt to analyze candidate genes, genetic variation was assessed in three cytochrome P-450 genes involved in the metabolism of cyclophosphamide. Resequencing of eight strains identified 26 polymorphisms in these genes that may influence response to cyclophosphamide. Distinct regions of high- and low-polymorphism rates were identified, and two common haplotypes were shared among the strains for each gene that exhibited variation. This phenotypic and genotypic variation among inbred strains provides a framework for cyclophosphamide pharmacogenomic discovery.

Inhibition of the protective effect of cyclophosphamide by pre-treatment with buthionine sulfoximine.

Low dose cyclophosphamide (CTX) is protective against a subsequent challenge with a lethal dose of the same drug administered 5 days later. At the time of maximal protection, elevation of glutathione (GSH) and glutathione transferase (GST) levels are detectable in the bone marrow of pre-treated animals. Elevation of GSH levels in the bone marrow was inhibited with the use of D,L-buthionine-S,R-sulfoximine (BSO), and this resulted in loss of the protective effect of CTX pre-treatment. In contrast, the overshoot in GST levels observed in these animals was not affected by BSO therapy. Bone marrow GSH levels in animals treated with BSO alone were minimally depleted (68% of control); whereas, animals pre-treated with CTX followed by BSO exhibited a greater reduction in GSH levels (47% of control). These results suggest that GSH is important in the protective effect afforded by low dose CTX pre-treatment and that the elevation of GSH levels observed is the result of a rebound synthetic process. In CTX pre-treated animals, BSO treatment resulted in greater than predicted depletion in GSH levels, and, therefore, caution is recommended with the potential use of combinations of BSO and cytotoxic drugs in the presence of a regenerating bone marrow.

Interference in food aversion conditioning by reducing drug dose or conditioning trials.

Previous work has suggested that aversions to foods are relatively resistant to interference effects. Although these findings have been interpreted as reflecting the potency of foods as targets in aversion conditioning, resistance to interference could more generally characterize intense conditioned aversions. In the present studies with Wistar rats we examined whether interference with food aversion conditioning could be demonstrated by reducing either the drug dose (cyclophosphamide; 20 vs 40 mg/kg) or the number of conditioning trials. In both cases interference was produced; that is, the presence of a second food did interfere with the conditioning of aversions to the first or "target" food. Thus, under conditions which would be expected to produce slower or weaker conditioning, interference with food aversions was facilitated.

One-trial long-lasting food-aversion learning in wild Japanese monkeys (Macaca fuscata).

We examined how Japanese monkeys in the wild formed an aversion to food which had been paired with poison. Ten monkeys of various ages and both sexes were chosen as subjects from 105 members of the Shiga-A1 troop at Jigokudani in Shiga Heights in Japan. We gave almond nuts to each subject. Once a monkey ate 10-20 almond nuts, he was captured and moved into an injection cage. Seven experimental subjects were injected intravenously with cyclophosphamide (20 mg/kg). Three control subjects received the same treatment except that they were injected with physiological saline. About 1 hour later, all subjects were released into the troop. The tests for conditioned aversions were conducted during the next 2 days. In the tests, the experimental subjects would not eat almond nuts, while the control subjects showed no hesitation in eating them. Five of the seven experimental subjects retained perfectly the aversion to almond nuts in tests conducted 1 month and 3 months later. The one-trial long-lasting food-aversion learning shown by the wild Japanese monkeys is discussed in terms of their feeding strategy. These results also suggest that food-aversion conditioning has potential as a nonlethal method for controlling crop-raiding monkeys.

Relative potency of foods and drinks as targets in aversion conditioning.

The potency of food stimuli as targets in aversion learning was examined using an interference paradigm. In the first study, foods interfered with liquid aversions but liquids did not interfere with food aversions. In the second study, food aversions were found to be resistant to interference by other foods. These findings suggest that foods are relatively potent targets in aversion conditioning in that they are resistant to interference by both foods and drinks. The final study examined the contribution of flavor intensity and nutrient density to the potency of aversion conditioning. The relative potency of foods over drinks may reflect differences in their intensity as well as their different roles in toxin avoidance and nutrient selection.

Effects of interference stimuli on the acquisition of learned aversions to foods in the rat.

A learned food aversion paradigm was developed to investigate characteristics of aversion learning likely to be involved in nutrient selection. The development of aversions to a relatively familiar, complete food was examined in rats with continuous access to that food. Learned food aversions were observed which were specific to the particular diet present during a sequence of toxic drug injections. In subsequent studies, novel liquids and solids were introduced in association with drug treatment to examine their effectiveness as interference stimuli. Flavored solutions, which have previously been reported to interfere with conditioned taste aversions, had little or no effect on learned aversions to the diet. This lack of interference by novel flavored liquids was coupled with significant conditioned aversions to the novel liquids themselves. Novel flavored solutions were contrasted with novel foods, which were extremely effective as interference stimuli in this paradigm. Possible bases for the observed differences in interference effects of foods and drinks are examined.

Intensity coding in the transient portion of the rat chorda tympani response.

For assessment of the relative importance of the transient and steady state components of the chorda tympani response to intensity coding of gustatory stimuli, the rat's ability to discriminate aversive solutions following adaptation was compared with changes in the neural response also resulting from adaptation. A taste aversion was conditioned to .9% NaCl, and subsequent acceptance of several NaCl concentrations (range: .1%-1.5%) was tested following infusion of 1 ml of adapting solution (H2O or .1%, .9%, or 1.5% NaCl) through an intra-oral cannula. Adaptation significantly decreased gustatory intensity discrimination and decreased the magnitude of the transient portion of the chorda tympani response. The degree of reduction in transient magnitude correlated significantly with reduced discrimination in all adapting conditions. However, the magnitude of the steady state response negatively correlated with discrimination. These data suggest that gustatory intensity discrimination depends upon information contained in the transient portion of the neural response and that the transient is necessary as well as sufficient for taste intensity discrimination.

Conditioned taste aversions accompanied by geophagia: evidence for the occurrence of "psychological" factors in the etiology of pica.

A conditioned taste aversion procedure was used to produce an avoidance of saccharin by rats. In the first experiment, saccharin consumption was paired with cyclophosphamide in two conditioning trials during which the animals were permitted to engage in pica. In the second experiment, saccharin consumption was paired with lithium chloride in four conditioning trials during which the animals were not allowed to engage in pica. Conditioned animals in both experiments subsequently engaged in geophagia when presented with saccharin alone. The absence of geophagia in noncontingently poisoned and "sham" injected control groups indicates that the pica was due to the acquisition of a conditioned illness during the conditioning trials. In addition to providing a demonstration of "psychological" involvement in the etiology of pica, these results indicate that visceral conditioning may accompany the formation of conditioned taste aversions. It is suggested that if there is a relationship between infantile pica and adult drug addiction, a plausible mediational mechanism is that pica-prone and addiction-prone individuals are similar in possessing a high susceptibility to visceral conditioning.

Effects of lesions of the gustatory necortex on taste aversion learning in the rat.

Normal rats and rats with lesions of the gustatory neocortex (GN) were compared for their ability to learn aversions to taste cues paired with toxicosis. When the taste presentation was followed immediately by toxicosis, normal rats and rats with lesions of the posterior (visual) neocortex learned aversions to sucrose, sodium chloride, quinine hydrochloride, and hydrochloric acid solutions. Rats with GN lesions learned aversions to all solutions except sucrose. In preference tests, all solutions were shown to be discriminable from water by both normal rats and rats with GN lesions. Under conditions in which a 6-hr delay separated taste presentation and toxicosis, normal rats again learned specific aversions to all four solutions, but rats with GN lesions failed to learn specific aversions to sucrose, sodium chloride, and hydrochloric acid solutions. It was shown that the ability of rats with GN lesions to learn aversions to sucrose and quinine depended on stimulus concentration; and it was proposed that the data could be accounted for by postulating a change in the threshold for taste-illness associations following GN lesions.