Showing papers in "Journal of Applied Toxicology in 2001"

A good practice guide to the administration of substances and removal of blood, including routes and volumes

Abstract: This article is the result of an initiative between the European Federation of Pharmaceutical Industries Associations (EFPIA) and the European Centre for the Validation of Alternative Methods (ECVAM). Its objectives are to provide the researcher in the safety evaluation laboratory with an up-to-date, easy-to-use set of data sheets to aid in the study design process whilst at the same time affording maximum welfare considerations to the experimental animals. Although this article is targeted at researchers in the European Pharmaceutical Industry, it is considered that the principles underpinning the data sets and refinement proposals are equally applicable to all those who use these techniques on animals in their research, whether in research institutes, universities or other sectors of industry. The implications of this article may lead to discussion with regulators, such as those responsible for pharmacopoeial testing. There are numerous publications dealing with the administration of test substances and the removal of blood samples, and many laboratories also have their own "in-house" guidelines that have been developed by custom and practice over many years. Within European Union Directive 86/609EEC1 we have an obligation to refine experiments to cause the minimum amount of stress. We hope that this article will provide background data useful to those responsible for protocol design and review. This guide is based on peer-reviewed publications whenever possible, but where this is not possible we have used "in-house" data and the experience of those on the working party (as well as helpful comments submitted by the industry) for a final opinion. The guide also addresses the continuing need to refine the techniques associated with the administration of substances and the withdrawal of blood, and suggests ways of doing so. Data-sharing between laboratories should be encouraged to avoid duplication of animal work, as well as sharing practical skills concerning animal welfare and scientific problems caused by "overdosing" in some way or another. The recommendations in this guide refer to the "normal" animal, and special consideration is needed, for instance, during pregnancy and lactation. Interpretation of studies may be confounded when large volumes are administered or excessive sampling employed, particularly if anaesthetics are used.

The glutathione defense system in the pathogenesis of rheumatoid arthritis.

Abstract: In order to assess a possible role of the natural glutathione defense system in the pathogenesis of rheumatoid arthritis (RA), serum reduced glutathione levels (GSH), glutathione reductase (GSR), glutathione S-transferase (GST), glutathione peroxidase (GSH-Px) and alkaline phosphatase (ALP) activities, lipid peroxidation (MDA content) and indexes of inflammation were evaluated in 58 rheumatic patients. Rheumatoid athritis was associated with significant depletion (ca. 50%) in GSH levels compared with normal control subjects. Serum levels of the detoxifying enzymes GSR and GSH-Px decreased by ca. 50% and 45%, respectively, whereas a threefold increase in the activity of GST was observed. A 1.2-fold increase in ALP was observed in patients with RA. These effects were accompanied by a 3.1-fold increase in serum MDA content. The MDA content was higher in RA patients who were seropositive for rheumatoid factor as well as positive for C-reactive proteins. The erythrocyte sedimentation rate for all patients with RA was approximately 13.8-fold higher than for the control group, and was higher among RA patients who were positive for C-reactive proteins and exhibited seropositivity for rheumatoid factor. Patients with RA receiving gold therapy exhibited significantly lower MDA levels whereas all other factors that were measured were not effected. The results support a hypothesis that defense mechanisms against reactive oxygen species are impaired in RA. Copyright © 2001 John Wiley & Sons, Ltd.

Riot control agents: pharmacology, toxicology, biochemistry and chemistry

Abstract: The desired effect of all riot control agents is the temporary disablement of individuals by way of intense irritation of the mucous membranes and skin. Generally, riot control agents can produce acute site-specific toxicity where sensory irritation occurs. Early riot control agents, namely, chloroacetophenone (CN) and chlorodihydrophenarsazine (DM), have been replaced with ‘safer’ agents such as o-chlorobenzylidene malononitrile (CS) and oleoresin of capsicum (OC). Riot control agents are safe when used as intended: however, the widespread use of riot control agents raises questions and concerns regarding their health effects and safety. A large margin exists between dosages that produce harassment and dosages likely to cause adverse health effects for modern riot control agents such as CS and dibenz[b,f]1 : 4-oxazepine (CR). Yet, despite the low toxicity of modern riot control agents, these compounds are not entirely without risk. The risk of toxicity increases with higher exposure levels and prolonged exposure durations. Ocular, pulmonary and dermal injury may occur on exposure to high levels of these substances, and exposure to riot control agents in enclosed spaces may produce significant toxic effects. Reported deaths are few involving riot control agents, and then only under conditions of prolonged exposure and high concentrations. Recently, concern has focused on the deaths resulting from law enforcement use of OC, a riot control agent generally regarded as safe because it is a natural product. As with other xenobiotics, not enough is known concerning the long-term/chronic effects of riot control agents. Clearly, there is considerable need for additional research to define and delineate the biological and toxicological actions of riot control agents and to illuminate the full health consequences of these compounds as riot control agents. Copyright © 2001 John Wiley & Sons, Ltd.

Chronic toxicity/oncogenicity study of styrene in CD-1 mice by inhalation exposure for 104 weeks

Abstract: Groups of 70 male and 70 female Charles River CD-1 mice were exposed whole body to styrene vapor at 0, 20, 40, 80 or 160 ppm 6 h per day 5 days per week for 98 weeks (females) or 104 weeks (males). The mice were observed daily; body weights, food and water consumption were measured periodically, a battery of hematological and clinical pathology examinations were conducted at weeks 13, 26, 52, 78 and 98 (females)/104 (males). Ten mice of each gender per group were pre-selected for necropsy after 52 and 78 weeks of exposure and the survivors of the remaining 50 of each gender per group were necropsied after 98 or 104 weeks. An extensive set of organs from the control and high-exposure mice were examined histopathologically, whereas target organs, gross lesions and all masses were examined in all other groups. Styrene had no effect on survival in males. Two high-dose females died (acute liver toxicity) during the first 2 weeks; the remaining exposed females had a slightly higher survival than control mice. Levels of styrene and styrene oxide (SO) in the blood at the end of a 6 h exposure during week 74 were proportional to exposure concentration, except that at 20 ppm the SO level was below the limit of detection. There were no changes of toxicological significance in hematology, clinical chemistry, urinalysis or organ weights. Mice exposed to 80 or 160 ppm gained slightly less weight than the controls. Styrene-related non-neoplastic histopathological changes were found only in the nasal passages and lungs. In the nasal passages of males and females at all exposure concentrations, the changes included respiratory metaplasia of the olfactory epithelium with changes in the underlying Bowman's gland; the severity increased with styrene concentration and duration of exposure. Loss of olfactory nerve fibers was seen in mice exposed to 40, 80 or 160 ppm. In the lungs, there was decreased eosinophilia of Clara cells in the terminal bronchioles and bronchiolar epithelial hyperplasia extending into alveolar ducts. Increased tumor incidence occurred only in the lung. The incidence of bronchioloalveolar adenomas was significantly increased in males exposed to 40, 80 or 160 ppm and in females exposed to 20, 40 and 160 ppm. The increase was seen only after 24 months. In females exposed to 160 ppm, the incidence of bronchiolo-alveolar carcinomas after 24 months was significantly greater than in the controls. No difference in lung tumors between control and styrene-exposed mice was seen in the intensity or degree of immunostaining, the location of tumors relative to bronchioles or histological type (papillary, solid or mixed). It appears that styrene induces an increase in the number of lung tumors seen spontaneously in CD-1 mice.

Biomarkers as biological indicators of xenobiotic exposure.

Abstract: The presence of a xenobiotic in the environment always represents a risk for living organisms. However, to talk about impregnation there is a need to detect toxicity in the organism, and the concept of intoxication is related to specific organ alterations and clinical symptoms. Moreover, the relationship between the toxic levels within the organism and the toxic response is rather complex and has a difficult forecast because it depends on several factors, namely toxicokinetic and genetic factors. One of the methods to quantify the interaction with xenobiotics and its potential impact on living organisms, including the human being, is monitoring by the use of the so-called biomarkers. They can provide measures of the exposure, toxic effect and individual susceptibility to environmental chemical compounds and may be very useful to assess and control the risk of long-term outcomes associated with exposure to xenobiotic (i.e. heavy metals, halogenated hydrocarbons, pesticides).

Toxicology update: the cardiotoxicity of the oxidative stress metabolites of catecholamines (aminochromes).

Abstract: This toxicology update reviews the oxidative stress metabolites of catecholamines, postulated to be the biochemical initiators of cardiotoxicity. A brief overview of catecholamine metabolism is provided with several noteworthy historical observations relating to the autoxidation and rearrangement of epinephrine. The basic chemical and physical properties of adrenochrome and adrenolutin are discussed. The autoxidative, enzymatic and cellular basis for the transformation of catecholamines to oxidative metabolites is reviewed. Mechanisms seeking to account for the observed cardiotoxic changes in isolated heart perfusion studies and in vivo models are described.

Toxicological, medical and industrial hygiene aspects of glutaraldehyde with particular reference to its biocidal use in cold sterilization procedures.

Abstract: Aqueous solutions of > or =5% glutaraldehyde (GA) are of moderate acute peroral toxicity and those of or =45% are of moderate acute percutaneous toxicity, those of 25% are of slight toxicity and those of or =5%. Primary skin irritation depends on the duration and contact site, occlusion and solvent. By sustained contact, the threshold for skin irritation is 1%, above which erythema and edema are dose related. With 45% and higher, skin corrosion may occur. There is a low incidence of skin sensitizing reactions, with an eliciting threshold of 0.5% aqueous GA. However, GA is neither phototoxic nor photosensitizing. Subchronic repeated exposure studies by the peroral route show only renal physiological compensatory effects, secondary to reduced water consumption. Repeated skin contact shows only minor skin irritant effects without systemic toxicity. By subchronic vapor exposure, effects are limited to the nasal mucosa at 1.0 ppm, with a no-effect concentration generally at 0.1 ppm. There is no evidence for systemic target organ or tissue toxicity by subchronic repeated exposure by any route. A chronic drinking water study showed an apparent increase, in females only, of large granular cell lymphocytic leukemia but this was not dosage related. This is most likely the result of a modifying effect on the factor(s) responsible for the expression of this commonly occurring rat neoplasm. A chronic (2-year) inhalation toxicity/oncogenicity study showed inflammatory changes in the anterior nasal cavity but no neoplasms or systemic toxicity. In vitro genotoxicity studies--bacterial mutagenicity, forward gene mutation (HGPRT and TK loci), sister chromatid exchange, chromosome aberration, UDS and DNA repair tests--have given variable results, ranging from no effect through to weak positive. In vivo genotoxicity studies--micronucleus, chromosome aberration, dominant lethal and Drosophila tests--generally have shown no activity but one mouse intraperitoneal study showed bone marrow cell chromosome aberrations. Developmental toxicity studies show GA not to be teratogenic, and a two-generation study showed no adverse reproductive effects. Percutaneous pharmacokinetic studies showed low skin penetration, with lowest values measured in vitro in rats and human skin. Overexposure of humans produces typical sensory irritant effects on the eye, skin and respiratory tract. Some reports have described an asthmatic-like reaction by overexposure to GA vapor. In most cases this resembles reactive airways dysfunction syndrome, and the role of immune mechanisms is uncertain. Local mucosal effects may occur if medical instruments or endoscopes are not adequately decontaminated. Protection of individuals from the potential adverse effects of GA exposure requires that there be adequate protection of the skin, eyes and respiratory tract. The airborne concentration of GA vapor should be kept below the recommended safe exposure level (e.g. the threshold limit value) by the use of engineering controls. Those who work with GA should, through a training program, be aware of the properties of GA, its potential adverse effects, how to handle the material safely and how to deal with accidental situations involving GA. If effects develop in exposed workers, the reasons should be determined immediately and corrective methods initiated. (c) 2001 John Wiley & Sons, Ltd.

Potential protective role of angiotensin-converting enzyme inhibitors captopril and enalapril against adriamycin-induced acute cardiac and hepatic toxicity in rats.

Abstract: Captopril and enalapril-angiotensin-converting enzyme (ACE) inhibitors-were evaluated for their antioxidative protective action against adriamycin-induced cardiac and hepatic toxicity. Rats were treated with either captopril (10 mg kg(-1)) or enalapril (2 mg kg(-1)) intragastrically (i.g.) daily for 7 days before single intraperitoneal (i.p.) injection with adriamycin (15 mg kg(-1)). The animals were killed 30 h after adriamycin administration. Adriamycin produced significant elevation in thiobarbituric acid reactive substances (TBARS), which is an indicator of lipid peroxidation, and significantly inhibited the activity of superoxide dismutase (SOD) in heart and liver tissues, with a significant rise in the serum levels of glutamic pyruvic transaminase (GPT), glutamic oxaloacetic transaminase (GOT), creatine kinase isoenzyme (CK-MB) and lactic dehydrogenase (LDH), indicating acute cardiac toxicity. A single injection of adriamycin did not affect the cardiac or hepatic glutathione (GSH) content or cardiac catalase (CAT) activity, but hepatic CAT activity was elevated. Pretreatment with ACE inhibitors significantly reduced the TBARS concentration in both heart and liver and ameliorated the inhibition of cardiac and hepatic SOD activity. In addition, the ACE inhibitors significantly improved the serum levels of GOT, GPT, CK-MB and LDH in adriamycin-treated rats. Thus, these results suggest that captopril and enalapril possess antioxidative potential that may protect the heart against adriamycin-induced acute oxidative toxicity. This protective effect might be mediated, at least in part, by the limitation of culprit free radicals and the amelioration of oxidative stress.

Review on the toxicity of ethylmercury, including its presence as a preservative in biological and pharmaceutical products

Abstract: Chemicals/CAS: Biological Products; Ethylmercury Compounds; Food Additives; Pharmaceutical Preparations; Preservatives, Pharmaceutical; Thimerosal, 54-64-8

Carboxylesterase: specificity and spontaneous reactivation of an endogenous scavenger for organophosphorus compounds

Abstract: The ability of carboxylesterase (CaE) to act as a bioscavenger to provide protection against organophosphorus (OP) compounds has been demonstrated in several animal models. To further evaluate the effectiveness of CaE as a bioscavenger, the specificity and stoichiometry of the detoxication of OP compounds by rat plasma CaE were examined. The specificity of CaE was evaluated by determining the bimolecular rate constants for inhibition (k(i)) of CaE by a variety of OP compounds. CaE exhibited a broad specificity for neutral OP compounds with k(i) > 10(6) M(-1) x min(-1) for paraoxon, sarin, soman, diisopropyl fluorophosphate, and diphenyl p-nitrophenyl phosphinate. CaE exhibited poor reactivity (k(i) sarin > soman). The pH dependence of the spontaneous reactivation of sarin-inhibited CaE suggested that its reactivation was dependent on an amino acid residue with a pK(a) of 6.1, which is probably a histidine that is highly conserved in CaE but not in other esterases.

The active site of human paraoxonase (PON1)

Abstract: Ideally we would like to treat people exposed to nerve agents with an enzyme that rapidly destroys nerve agents. The enzymes considered for such a role include human butyrylcholinesterase (BChE), acetylcholinesterase (AChE), carboxylesterase and paraoxonase (PON1). Success has been achieved in endowing BChE with the ability to hydrolyze organophosphates. The G117H mutant of BCHE hydrolyzes sarin and VX, whereas the double mutant G117H/E197Q hydrolyzes soman (Millard et al. Biochemistry 1995; 34: 15925-15933; 1998; 37: 237-247). However, the rates of organophosphate hydrolysis are slow and a faster organophosphate hydrolase is being sought. Native PON1 hydrolyzes paraoxon with a catalytic efficiency, of 2.4 x 10(6) M(-1) x min(-1), and our goal is to improve the organophosphate hydrolase activity of PON1. To achieve this we need to identify the amino acids in the active site of PON1. Using site-directed mutagenesis and expression in human 293T cells, we have identified the following eight amino acids as being essential to PON1 activity: W280, H114, H133, H154, H242, H284, E52 and D53. Fluorescence of PON1 complexed to terbium ion shows that at least one tryptophan is close to the calcium binding site.

N-acetylcysteine and endothelial cell injury by sulfur mustard.

Abstract: Understanding the underlying mechanisms of cell injury and death induced by the chemical warfare vesicant sulfur mustard (HD) will be extremely helpful in the development of effective countermeasures to this weapon of terror. We have found recently that HD induces both apoptosis and necrosis in endothelial cells (Toxicol. Appl. Pharmacol. 1996; 141: 568-583). Pretreatment of the endothelial cells for 20 h with the redox-active agent N-acetyl-L-cysteine (NAC) selectively prevented apoptotic death induced by HD. In this study, we tested the hypotheses that pretreatment with NAC acts through two different pathways to minimize endothelial injury by HD: NAC pretreatment acts via a glutathione (GSH)-dependent pathway; and NAC pretreatment acts to suppress HD-induced activation of the nuclear transcription factor NFkappaB. We used a fluorescence microscopic assay of apoptotic nuclear features to assess viability and electrophoretic mobility shift assays (EMSAs) to assess the activity of NFkappaB following exposure to HD. The cells were treated with 0-10 mM GSH for 1 h prior to and during exposure to 0 or 500 microM HD for 5-6 h. Cells were also treated with 50 mM NAC or 200 microM buthionine sulfoximine (BSO), an inhibitor of GSH synthesis, alone or in combination overnight prior to exposure to 0 or 500 microM HD for 5-6 h. Externally applied GSH up to a concentration of 5 mM had no toxic effect on the cells. Mild toxicity was associated with 10 mM GSH alone. There was a dose-related enhancement of viability when 2.5 and 5 mM GSH were present during the HD exposure. Pretreatment with BSO alone had no discernible toxicity. However, pretreatment with this inhibitor of GSH synthesis potentiated the toxicity of HD. Pretreatment with 50 mM NAC, as previously reported, provided substantial protection. Combining pretreatment with both BSO and NAC eliminated the protective effect of NAC pretreatment alone on HD injury. These observations are highly suggestive that NAC enhances endothelial survival via GSH-dependent effects and confirms and extends the work of others with different models that externally supplied GSH alone may be a fairly effective countermeasure against HD injury of endothelium. We next examined the hypothesis that HD may activate the nuclear transcription factor NFkappaB by performing EMSAs with nuclear extracts of endothelial cells following exposure to 0, 250 or 500 microM HD. This demonstrated an up to 2.5-fold increase (scanning densitometry) in activation of NFkappaB binding to its consensus sequence induced by 500 microM HD after 5 h of HD exposure. Paradoxically, treatment of the endothelial cells alone with 50 mM NAC activated NFkappaB, although HD-induced activation of NFkappaB was partially suppressed by NAC at 5 h. Factor NFkappaB is an important transcription factor for a number of cytokine genes (e.g. tumor necrosis factor, TNF), which can be activated following stress in endothelial cells. Taken together, these observations suggest that the protective effects of NAC may be mediated by enhanced GSH synthesis. The increased GSH may act to scavenge HD and also prevent oxidative activation of NFkappaB. Under some conditions, NAC may act as an oxidizing agent and thus increase NFkappaB activity. The NFkappaB-dependent gene expression may be important in inducing endothelial cell death as well as in generating a local inflammatory reaction associated with the release of endothelial-derived cytokines.

Improved erythrocyte lysis assay in microtitre plates for sensitive detection and efficient measurement of haemolytic compounds from ichthyotoxic algae

Abstract: Haemolytic substances produced by ichthyotoxic algae often are unknown in molecular structure or specific mechanism of toxicity. Detection and quantification of such substances are dependent on bioassays, using markers that are sensitive for haemolytic impairment and generation of a recordable response. The erythrocyte lysis assay (ELA) represents an advantageous bioassay in this respect, because the lytic response can be measured photometrically by the amount of released haemoglobin. The aim of the present study was to establish an improved assay based on the ELA principle, for sensitive determination of haemolytic substances of microalgae and for high sample throughput. For this purpose we adapted the ELA to a 96-well microtitre plate format, which significantly reduced the sample volumes and allowed rapid processing of samples. Further improvement was achieved by measuring absorption of lysed erythrocytes at 414 nm, which significantly increased the sensitivity of the ELA compared to the measurements at 540 nm that are usually applied in this type of assay. Using carp (Cyprinus carpio) erythrocytes it was possible to detect haemolysis induced by 4 microg ml(-1) of saponin and as little as two haemolytic Alexandrium tamarense cells. It is suggested that this improved ELA in microtitre plates be used as a low-cost monitoring tool for detection and analysis of potential harmful algae. Furthermore, this ELA can be useful as a sensitive screening system for substances of pharmacological interest, e.g. selectively acting cytolytic antibiotics.

The NMDA receptor ion channel: a site for binding of Huperzine A.

Abstract: Huperzine A (HUP-A), first isolated from the Chinese club moss Huperzia serrata, is a potent, reversible and selective inhibitor of acetylcholinesterase (AChE) over butyrylcholinesterase (BChE) (Life Sci. 54: 991-997). Because HUP-A has been shown to penetrate the blood-brain barrier, is more stable than the carbamates used as pretreatments for organophosphate poisoning (OP) and the HUP-A:AChE complex has a longer half-life than other prophylactic sequestering agents, HUP-A has been proposed as a pretreatment drug for nerve agent toxicity by protecting AChE from irreversible OP-induced phosphonylation. More recently (NeuroReport 8: 963-968), pretreatment of embryonic neuronal cultures with HUP-A reduced glutamate-induced cell death and also decreased glutamate-induced calcium mobilization. These results suggest that HUP-A might interfere with and be beneficial for excitatory amino acid overstimulation, such as seen in ischemia, where persistent elevation of internal calcium levels by activation of the N-methyl-D-aspartate (NMDA) glutamate subtype receptor is found. We have now investigated the interaction of HUP-A with glutamate receptors. Freshly frozen cortex or synaptic plasma membranes were used, providing 60-90% specific radioligand binding. Huperzine A (< or =100 microM) had no effect on the binding of [3H]glutamate (low- and high-affinity glutamate sites), [3H]MDL 105,519 (NMDA glycine regulatory site), [3H]ifenprodil (NMDA polyamine site) or [3H]CGS 19755 (NMDA antagonist). In contrast with these results, HUP-A non-competitively (Hill slope < 1) inhibited [3H]MK-801 and [3H]TCP binding (co-located NMDA ion channel PCP site) with pseudo K(i) approximately 6 microM. Furthermore, when neuronal cultures were pretreated with HUP-A for 45 min prior to NMDA exposure, HUP-A dose-dependently inhibited the NMDA-induced toxicity. Although HUP-A has been implicated to interact with cholinergic receptors, it was without effect at 100 microM on muscarinic (measured by inhibition of [3H]QNB or [3H]NMS binding) or nicotinic [3H]epibatidine binding) receptors; also, HUP-A did not perturb adenosine receptor binding [3H]PIA or [3H]NECA). Therefore, HUP-A most likely attenuates excitatory amino acid toxicity by blocking the NMDA ion channel and subsequent Ca2+ mobilization at or near the PCP and MK-801 ligand sites. Thus, on the one hand, HUP-A could be used as a pretreatment against OPs and it might also be a valuable therapeutic intervention in a variety of acute and chronic disorders by protecting against overstimulation of the excitatory amino acid pathway. By blocking NMDA ion channels without psychotomimetic side-effects, HUP-A may protect against diverse neurodegenerative states observed during ischemia or Alzheimer's disease.

Influence of lead and zinc on rat male reproduction at 'biochemical and histopathological levels'.

Abstract: Sequential lead accumulation and biochemical and histopathological changes were observed in rat testis and epididymis after oral administration at varied doses of lead (10, 50 and 200 mg kg(-1) body wt.) for 3 months and also following the concomitant administration of lead with zinc (1 mg kg(-1) body wt. +50 mg Pb kg(-1) body wt.). Accumulation of lead in both testis and epididymis increased with dose. The concomitant administration of zinc reduced the lead levels. Similarly, dose-related changes were seen in the activities of the enzymes alkaline phosphatase and Na(+)-K(+)-ATPase, which decreased with increased dose of lead. A significant improvement in the activities of these enzymes was seen in the groups given both lead and zinc. Histologically, discernible changes were noticed only at higher doses (50 and 200 mg kg(-1) body wt.), which included disorganization and disruption of spermatogenesis with accumulation of immature cells in lumen of tubule. At higher doses of lead, complete arrest of spermatogenesis was seen and a significant decrease in germ cell layer population was evident. Even in epididymis, the histoarchitecture was disrupted only at higher doses of lead both in the caput and corpus regions. The changes included damage of basement membrane, disorganization of epithelium and vacuolization of cells. The tubules were found almost empty, indicating arrest of spermatogenesis. However, with concomitant administration of lead and zinc both testis and epididymis presented a near-normal picture, indicating the protective role of zinc. Hence, the data indicate that the protective effect of zinc on lead toxicity was mediated largely by significant competition between lead and zinc or due to reduction of the available binding sites.

Therapeutic approaches to dermatotoxicity by sulfur mustard I. Modulation of sulfur mustard‐induced cutaneous injury in the mouse ear vesicant model†**

Abstract: The mouse ear edema model is recognized for its usefulness in studying skin responses and damage following exposure to chemical irritants, and for evaluating pharmacological agents against chemically induced skin injury. We recently modified the mouse ear edema model for use with sulfur mustard (HD) and used this model to study the protective effect of 33 topically applied compounds comprising five pharmaceutical strategies (anti-inflammatories, protease inhibitors, scavengers/chelators, poly(ADP-ribose) polymerase (PARP) inhibitors, calcium modulators/chelators) against HD-induced dermatotoxicity. Pharmacological modulation of HD injury in mouse ears was established by a reduction in edema or histopathology (epidermal necrosis and epidermal-dermal separation) at 24 h following topical liquid HD exposure. Ten of the 33 compounds administered as single topical pretreatments up to 2 h prior to HD challenge produced significant reductions in edema. Five of these ten also produced significant reductions in histological endpoints. Three candidates (olvanil, indomethacin, hydrocortisone) showing protection at 24 h were evaluated further for 'extended protection' at 48 and 72 h after HD challenge and showed significant modulation of edema at 48 h but not at 72 h. Olvanil also showed significant reductions in histology at 48 and 72 h. Olvanil and indomethacin were shown to reduce significantly the edema at 24 h post-exposure when administered topically 10 min after HD challenge, with olvanil additionally protecting against epidermal necrosis. These results demonstrate prophylactic and treatment effects of pharmacological agents against HD-induced skin injury in an in vivo model and support the continued use of the mouse ear vesicant model (MEVM) for evaluating medical countermeasures against HD.

Inflammatory cytokine response in sulfur mustard-exposed mouse skin.

Abstract: Assessment of anti-inflammatory therapies against sulfur-mustard (bis(2-chloroethyl)sulfide, HD)-induced skin injury has mainly relied on qualitative histopathological evaluation. Development of quantifiable inflammatory biomarkers using fast and reliable molecular methods is needed for screening anti-inflammatory drugs against HD injury. In this study, we used two different HD exposure models to determine the in vivo cutaneous response of the inflammatory cytokines interleukin-6 (IL-6), IL-1alpha, IL-1beta and tumor necrosis factor alpha (TNF-alpha), in order to identify a suitable inflammatory biomarker common to both models. In the first model, the backs of hairless mice were exposed to HD vapor (1.4 g m(-3)) or sham controls for 6 min using an occluded vapor cup technique. In the second model, right ears of CD1 mice were exposed to a solution (5.0 microl of 195 mM) of HD (0.16 mg) in dichloromethane (CH2Cl2) whereas left ears received only CH2Cl2 (vehicle control). Sulfur-mustard-induced skin inflammation was assessed in skin punch specimens collected at time points up to 24 h post-exposure. Edema was determined by measuring tissue weight, and cytokine content was measured by enzyme immunosorbent assay. Characterized by an increase in edema and IL-6, HD provoked a cutaneous inflammatory response in both models beginning at 6 h post-exposure and continuing to 24 h. An increase in IL-1alpha was observed only in the hairless mouse model, also beginning at 6 h post-exposure and continuing to 24 h. No IL-1beta or TNF-alpha response was observed at any time point in either exposure model. These data document the in vivo production of cutaneous IL-6, a distinct inflammatory biomarker, in two different HD exposure models. We conclude that IL-6 should be a useful in vivo biomarker for evaluating anti-inflammatory drugs against HD-induced skin injury.

Cytokine fingerprinting and hazard assessment of chemical respiratory allergy.

Abstract: Allergic sensitization of the respiratory tract resulting in occupational asthma and other symptoms can be caused by a variety of chemicals and represents an important occupational health problem. Although there is a need to identify and characterize those chemicals that are able to cause respiratory allergy, there are currently no well validated or widely accepted predictive test methods. Some progress has been made with guinea pig assays, but our attention in this laboratory has focused instead on the development of novel approaches based on an understanding of the nature of immune responses induced in mice by chemical allergens. We have shown that whereas contact allergens provoke in mice selective type 1 immune responses, characterized by the secretion by draining lymph node cells (LNC) of high levels of the cytokine interferon gamma (IFN-gamma), chemical respiratory allergens stimulate instead preferential type 2 responses associated with comparatively high levels of interleukins 4 and 10 (IL-4 and IL-10). The divergent immune responses provoked by different classes of chemical allergens, and the phenotypes of selective cytokine secretion that characterize such responses, form the basis of a novel method-cytokine fingerprinting--that permits chemicals that have the potential to cause respiratory allergy to be identified and distinguished from those that are associated primarily with contact sensitization. In this article the immunobiological basis for cytokine fingerprinting is considered and the development, evaluation and practical application of the assay are reviewed.

Response of normal human keratinocytes to sulfur mustard: cytokine release.

Abstract: Cytokines play a major role in both acute and chronic inflammatory processes, including those produced by sulfur mustard (2,2'-dichlorodiethyl sulfide, HD). This study describes responses of normal human epidermal keratinocytes (NHEK) to HD, defined by interleukin-1beta (IL-1beta), IL-6, IL-8 and tumor necrosis factor alpha (TNF-alpha) release. Commercially available enzyme-linked immunosorbent assay (ELISA) kits were used to measure the cytokine release in NHEK during exposure to 100 and 300 microM of HD. Exposure to 100 microM HD increased the release of cytokines. The amounts of IL-8 and TNF-alpha present in cell suspensions increased up to 59-fold and 4-fold, respectively, above control levels when NHEK were exposed to 300 microM HD. Exposure of NHEK to 300 microM HD had a highly variable effect on the release of IL-1beta, where sometimes the secretion of IL-1beta increased above baseline level and at other times it decreased in cell suspensions. Supernatants were collected from cell culture flasks 24 h after exposure of 100 and 300 microM HD and significantly increased levels of IL-6 were observed. Interleukin-6 was released in a concentration-dependent manner, 3.6-fold up to 8.4-fold, respectively, in supernatant. These pro-inflammatory mediators IL-1beta, IL-8, TNF-alpha and IL-6 may play an important role in HD injury. The present findings suggest that the cytokine changes detected could be used as potential biomarkers of cutaneous vesicant injury.

Production of superoxide anion, lipid peroxidation and DNA damage in the hepatic and brain tissues of rats after subchronic exposure to mixtures of TCDD and its congeners.

Abstract: In this study the induction of oxidative stress in the hepatic and brain tissues of rats after subchronic exposure to various mixtures of 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) and two of its congeners, namely 2,3,4,7,8-pentachlorodibenzofuran (PeCDF) and 3,3',4,4',5-pentachlorobiphenyl (PCB 126) was investigated. Four mixtures of TCDD and its congeners, corresponding to 10, 22, 46 and 100 ng of toxic equivalence (TEQ) kg(-1) day(-1), were administered to groups of rats for 13 weeks. The animals were sacrificed at the end of the exposure period and the biomarkers of oxidative stress, including the production of superoxide anion, lipid peroxidation and DNA single-strand breaks (SSBs), were determined in the hepatic and brain tissues. All mixtures caused dose-dependent increases in the production of superoxide anion, lipid peroxidation and DNA SSBs in both tissues, with significantly higher damage in the hepatic compared with the brain tissues. The 22 ng TEQ dose level (TEQ = 22) contains TCDD, PeCDF and PCB 126 at levels that correspond to 7.3, 14.5 and 73.3 ng kg(-1) day(-1), respectively, and it produced effects that correspond to ca. 50% of the maximal production of superoxide anion, lipid peroxidation and DNA SSBs in the hepatic and brain tissues of those animals. Relative to the doses that are required to produce 50% of the maximal production of the biomarkers of oxidative stress by the individual congeners in hepatic and brain tissues of rats, the concentrations of the congeners in TEQ = 22 did result in significant interactivity, probably in the form of additive effects in the hepatic but not in brain tissues.

Treatment of sulfur mustard (HD)-induced lung injury.

Abstract: An in vivo sulfur mustard (HD) vapor exposure model followed by bronchoalveolar lavage was developed previously in this laboratory to study biochemical indicators of HD-induced lung injury. This model was used to test two treatment compounds--niacinamide (NIA) and N-acetyl cysteine (NAC)--for their ability to ameliorate HD-induced biochemical changes. Anesthetized rats were intratracheally intubated and exposed to 0.35 mg of HD in 0.1 ml of ethanol or ethanol alone for 50 min. At the beginning of the exposure (t = 0), the rats were treated with either NIA (750 mg kg(-1)) or NAC (816 mg kg(-1)), i.p. At 24 h post-exposure, rats were euthanized and the lungs were lavaged with saline (three 5-ml washes). One milliliter of the recovered lavage fluid was analyzed for cellular components. The remaining fluid was centrifuged (10 min at 300 g) and the supernatant was assayed on a Cobas FARA clinical analyzer for lactate dehydrogenase (LDH), gamma-glutamyltransferase (GGT), albumin (ALB), total protein (TP) and glutathione peroxidase (GP). The HD alone and HD+NIA treatment caused significant increases in all of the biochemical parameters compared with control levels. The NAC treatment yielded LDH, ALB and TP values that, although elevated, were not significantly different from the control. The GP levels were significantly higher than the control but significantly lower than the HD alone levels, indicating some protection compared with the HD alone group. The GGT levels were unaffected by NAC compared with HD alone. Cytological analysis of lavage fluid showed that the percentages of neutrophils were 5.3 +/- 1.0 (mean +/- SEM) for control, 46.6 +/- 4.5 for HD, 31.4 +/- 4.7 for HD + NIA and 21.6 +/- 4.7 for HD + NAC, respectively. The neutrophil counts were significantly higher for the three HD-exposed groups vs controls; however, the NAC-treated group had neutrophil counts lower than HD alone, indicating decreased inflammatory response. These results show that NAC may be useful as a potential treatment compound for HD-induced lung injury.

Beneficial effects of topical anti-inflammatory drugs against sulfur mustard-induced ocular lesions in rabbits

Abstract: Ocular injuries following sulfur mustard (HD) exposure are characterized by an inflammatory response, observed as eyelid swelling, conjunctivitis, corneal oedema and cellular infiltration starting 1-4 h after exposure, depending on dose. These effects heal partially during the first 1-2 weeks after exposure, with the later appearance of neovascularization, recurrent erosions and recurrent oedema of the cornea (delayed response). We have shown previously that topically applied steroid treatment, administered after HD exposure, attenuated the extent of neovascularization, one of the characteristics of delayed ocular pathology in rabbits. The present study was designed to characterize further the initial inflammatory response and to elucidate the role of anti-inflammatory (AI) drugs as a potential therapy. Rabbit eyes were exposed to HD vapour (390 microg l(-1) for 2 min) and were treated with a topical commercial ophthalmic solution of dexamethasone or diclofenac, starting 1 h post-exposure (four times a day). Inflammation was evaluated by clinical observations, biochemical analysis of aqueous humour and by histology. Sulfur mustard exposure initiated typical clinical ocular symptoms within 4-6 h after exposure. Biochemical analysis of aqueous humour showed that protein content and prostaglandin E (PGE) increased significantly at 6 h and were still high 48 h after HD exposure. Light microscopy evaluation revealed severe damage to the cornea, characterized by epithelial denudation, oedema and cellular infiltration (mostly eosinophiles) in the stroma. Both treatments were effective in alleviating the clinical symptoms and in preventing the HD-induced increase in protein and PGE in the anterior chamber, as well as the cellular infiltration, in the corneal stroma. However, the AI treatments had no therapeutic effect on corneal erosions, and a short delay in epithelial regeneration was noted. It is concluded that AI drugs are potential candidates for the treatment of ocular lesions following HD exposure.

Soman-induced seizures: limbic activity, oxidative stress and neuroprotective proteins.

Abstract: Soman, a potent acetylcholinesterase inhibitor, induces status epilepticus in rats followed by conspicuous neuropathology, most prominent in piriform cortex and the CA3 region of the hippocampus. Cholinergic seizures originate in striatal–nigral pathways and with fast-acting agents (soman) rapidly spread to limbic related areas and finally culminate in a full-blown status epilepticus. This leads to neurochemical changes, some of which may be neuroprotective whereas others may cause brain damage. Pretreatment with lithium sensitizes the brain to cholinergic seizures. Likewise, other agents that increase limbic hyperactivity may sensitize the brain to cholinergic agents. The hyperactivity associated with the seizure state leads to an increase in intracellular calcium, cellular edema and metal delocalization producing an oxidative stress. These changes induce the synthesis of stress-related proteins such as heat shock proteins, metallothioneins and heme oxygenases. We show that soman-induced seizures cause a depletion in tissue glutathione and an increase in tissue ‘catalytic’ iron, metallothioneins and heme oxygenase-1. The oxidative stress induces the synthesis of stress-related proteins, which are indicators of ‘stress’ and possibly provide neuroprotection. These findings suggest that delocalization of iron may catalyze Fenton-like reactions, causing progressive cellular damage via free radical products. Copyright © 2001 John Wiley & Sons, Ltd.

Allergen-induced changes in interleukin 1 beta (IL-1 beta) mRNA expression by human blood-derived dendritic cells: inter-individual differences and relevance for sensitization testing.

Abstract: The development of in vitro methods for the identification of skin sensitizers based upon analysis of Langerhans cell (LC) function has been constrained by the fact that these cells represent only a minority population in the skin that, once isolated, alter their phenotype spontaneously and rapidly. Methods have been developed recently that allow the expansion in culture using appropriate cytokine conditions of LC-like dendritic cells (DCs) from certain tissues, including human peripheral blood. It has been demonstrated that culture of human blood-derived LC-like cells with selected potent contact allergens such as 2,4-dinitrofluorobenzene (DNFB) stimulates selective phenotypic changes, including the up-regulation of interleukin 1 beta (IL-1 beta) mRNA expression, under conditions where skin irritants are without effect. However, in our own previous investigations, we have observed that there appear to be differences between blood donors with respect to the responsiveness of DCs to DNFB-induced changes in IL-1 beta expression, differences that could compromise the utility of this approach as a screening method for contact allergens. We have therefore investigated donor variability in DC responsiveness to a panel of known human contact allergens (DNFB; paraphenylene diamine, PPD; methyl- chloroisothiazolinone/methylisothiazolinone, CMIT), to the skin irritant benzalkonium chloride and to the mitogen phorbol myristate acetate (PMA). Dendritic cells derived from all donors expressed IL-1 beta mRNA constitutively. Treatment of DCs isolated from donors with a responder phenotype to DNFB with PPD or CMIT resulted also in up-regulation of IL-1 beta mRNA expression, although such changes were always comparatively modest, generally resulting in a twofold induction compared with vehicle-treated controls. Dendritic cells derived from donors with a non-responder phenotype to DNFB failed also to respond to these additional contact allergens under conditions where the mitogen PMA caused similar increases in IL-1 beta expression to those observed for allergen-responsive donors. Benzalkonium chloride failed to provoke changes in the expression of this cytokine in any donor examined, irrespective of their responder phenotype. The temporal stability of the responder/non-responder DC phenotype was confirmed, with stable phenotypes with respect to DNFB-induced changes in IL-1 beta mRNA expression observed over a period of some 18 months. Fifty per cent (6/12) of donors tested over this period displayed a responder phenotype. These data demonstrate that chemical allergens do stimulate consistent changes in IL-1 beta mRNA expression in the proportion of donors who have a responsive phenotype, and that such responses are apparently selective for allergen using the relatively narrow range of materials assessed to date. However, the modest response to very strong contact allergens, coupled with the difficulties of responder/non-responder phenotypes, means that in its present form this approach does not lend itself to the routine assessment of skin sensitizing activity.

Lead-induced cell death in testes of young rats.

Abstract: Lead is a well-documented testicular toxicant. The present work was planned to study the occurrence of germ cell death after lead administration. Young growing rats were treated with 5, 10 and 20 mg kg−1 body weight of lead for 2 weeks. Cell death was assessed by employing in situ TUNEL staining, DNA electrophoresis and morphological examination of the tubules. The results showed that Pb induced significant numbers of germ cells to undergo apoptosis in the seminiferous tubules of rats treated with 20 mg kg−1 body weight. However, DNA fragmentation was not detected at any of the doses. The level of lead accumulation in the testis increased in a dose-dependent manner. Copyright © 2001 John Wiley & Sons, Ltd.

In vitro skin absorption and decontamination of sulphur mustard: comparison of human and pig-ear skin.

Abstract: The aim of this study was to evaluate the use of an in vitro skin diffusion cell system as a model for assessing decontaminants against the chemical warfare agent sulphur mustard (SM). The in vitro absorption rates of SM through heat-separated human (157 +/- 66 microg cm(-2) h(-1)) and pig-ear (411 +/- 175 microg cm(-2) h(-1)) epidermal membranes were in agreement with previous in vivo studies that quoted skin absorption rates of 150 and 366 microg cm(-2) h(-1), respectively. Decontaminants (fuller's earth, Ambergard and BDH spillage granules) were ranked in order of effectiveness by measuring the skin absorption rates and the percentage of applied dose of SM that penetrated human and pig-ear epidermal membranes. The effectiveness of fuller's earth measured in this in vitro study using human epidermal membranes was in agreement with a previous in vivo human volunteer study. Similarly, the effectiveness of fuller's earth and Ambergard measured in vitro with pig-ear epidermal membranes was in agreement with a previous in vivo study conducted on rats. However, there was complete disparity in the ranking of decontaminants between human and pig-ear epidermal membranes measured in vitro. Thus, although pig-ear skin may be a relatively good model for predicting the human skin absorption of SM, it is a poor model for testing decontamination systems. The results of this study further validate the use of Franz-type glass diffusion cells containing human epidermal membranes as a model for predicting in vivo human skin absorption.

Selective vulnerability to kainate-induced oxidative damage in different rat brain regions

Abstract: Some markers of oxidative injury were measured in different rat brain areas (hippocampus, cerebral cortex, striatum, hypothalamus, amygdala/piriform cortex and cerebellum) after the systemic administration of an excitotoxic dose of kainic acid (KA, 9 mg kg(-1) ip) at two different sampling times (24 and 48 h) Kainic acid was able to lower markedly (P < 005) the glutathione (GSH) levels in hippocampus, cerebellum and amygdala/piriform cortex (maximal reduction at 24 h) In a similar way, lipid peroxidation, as assessed by malonaldehyde and 4-hydroxyalkenal levels, significantly increased (P < 005) in hippocampus, cerebellum and amygdala/piriform cortex mainly at 24 h after KA In addition, hippocampal superoxide dismutase (SOD) activity decreased significantly (P < 005) with respect to basal levels by 24 h after KA application On the other hand, brain areas such as hypothalamus, striatum and cerebral cortex seem to be less susceptible to KA excitotoxicity According to these findings, the pattern of oxidative injury induced by systemically administered KA seems to be highly region-specific Further, our results have shown that a lower antioxidant status (GSH and SOD) seems not to play an important role in the selective vulnerability of certain brain regions because it correlates poorly with increases in markers of oxidative damage

Oxidative preconditioning affords protection against carbon tetrachloride-induced glycogen depletion and oxidative stress in rats.

Abstract: The rectal insufflation of a judicious dose of ozone, selected from that used in clinical practice, is able to promote oxidative preconditioning or oxidative stress tolerance preventing the hepatocellular damage mediated by free radicals. In order to evaluate the effects of ozone oxidative preconditioning on carbon tetrachloride-mediated hepatotoxicity, the following experimental protocol was designed: group 1 (negative control, sunflower oil i.p.); group 2 (CCl(4) in sunflower oil, 1 ml kg(-1) i.p.); group 3 (15 ozone-oxygen pretreatments at a dose of 1 mg kg(-1) via rectal insufflation + CCl(4) as in group 2); group 4 (ozone control group, 15 ozone-oxygen pretreatments + sunflower oil i.p.). Ozone pretreatment prevented glycogen depletion (as demonstrated by biochemical and histopathological findings) and avoided lactate overproduction associated with the hepatotoxic effects of CCl(4). The administration of CCl(4) increased lipid peroxidation (as measured by thiobarbituric acid-reactive substances) and uric acid levels and inhibited superoxide dismutase activity. All these deleterious effects induced by CCl(4) were prevented by ozone pretreatment. The administration of ozone without CCl(4) (ozone control group) did not produce any changes in the evaluated parameters. Our results showed that ozone treatment, in our experimental conditions, was able to prevent anaerobic glycolysis and oxidative stress induced by CCl(4).

Systemic administration of candidate antivesicants to protect against topically applied sulfur mustard in the mouse ear vesicant model (MEVM).

Abstract: The mouse ear vesicant model (MEVM) provides a quantitative edema response as well as histopathological and biochemical endpoints as measurements of inflammation and tissue damage following exposure to the chemical warfare agent sulfur mustard (HD). In the MEVM, several topically applied anti-inflammatory agents provided a significant degree of protection against HD-induced edema and dermal-epidermal separation. This study evaluated the protective effects of three of these pharmacological compounds when administered systemically in the MEVM. Alzet osmotic pumps were used to deliver a subcutaneous dose of the appropriate anti-inflammatory agent, starting 24 h before exposure to sulfur mustard and continuing until 24 h post-exposure to HD. Twenty-four hours after pump implantation, 5 μl of a 195 mM (0.16 mg) solution of sulfur mustard (density = 1.27 g ml -1 ; MW = 159; purity = 97.5%) in methylene chloride was applied to the inner surface of the right ear of each mouse. Sulfur mustard injury in the mouse ear was measured by both edema response (fluid accumulation) and histopathological damage (necrosis, epidermal-dermal separation). The systemic administration of hydrocortisone, indomethacin and olvanil provided a significant reduction in edema (24%, 26% and 22%, respectively) from the positive control. Compared to HD-positive controls, hydrocortisone, indomethacin and olvanil caused a significant reduction in subepidermal blisters (71%, 52% and 57%, respectively) whereas only hydrocortisone produced a significant reduction in contralateral epidermal necrosis (41%). We show here that these anti-inflammatory drugs are effective when administered systemically in the MEVM.

Structure-hepatoprotective activity relationship of 3,4-dihydroxycinnamic acid (caffeic acid) derivatives.

Abstract: 3,4-Dihydroxycinnamic acid (caffeic acid, CAF) is a natural product containing a catechol group with an alpha,beta-unsaturated carboxylic acid chain that has shown hepatoprotective properties. The aim of this work was to determine the importance of the 4-hydroxy, 3-hydroxy, 3,4-dihydroxy substituents and the double bond moiety on the hepatic pharmacological effects of the molecule. We compared the ability of the caffeic, 4-hydroxycinnamic, 3-hydroxycinnamic, cinnamic and 3,4-dihydroxyhydrocinnamic (a caffeic acid analogue without the double bond) acids at a dose of 50 mg kg(-1), p.o., to reduce the liver damage produced by CCl(4) (4 g kg(-1), p.o.) intoxication in the rat. Cinnamic acid, the non-hydroxylated analogue, only modestly protected the experimental animals challenged with CCl(4), suggesting that hydroxyl groups participate in the pharmacological properties of CAF. The 3,4-dihydroxyhydrocinnamic derivative did not show any significant differences when compared with the CAF effect in this model, suggesting that the double bond does not account for the liver pharmacological properties of CAF. In contrast, the 4-hydroxy substituent seems to be very important for hepatoprotective activity because the 4-hydroxy analogue improved almost every hepatic injury marker altered by CCl(4), and in a better way than CAF did.