Multiple Toxic Effects Research Articles

Analysis of iron, zinc, selenium and cadmium in paraffin-embedded prostate tissue specimens using inductively coupled plasma mass-spectrometry

Formalin-fixed paraffin-embedded (FFPE) tissue specimens represent a valuable and abundant resource of pathologic material for various biomedical studies. In the present study, we report the application of high-resolution inductively coupled mass-spectrometry (ICP-MS) for quantification of Fe, Zn, Se and Cd in FFPE prostate tissue. These elements have a possible role in the development of prostate diseases: while Zn and Se are needed for a healthy prostate, Cd shows multiple toxic and carcinogenic effects. Excessive accumulation of Fe induces the production of highly reactive hydroxyl radical species, which may play a role in cancer etiopathogenesis. To assess whether the levels of these metals in the FFPE prostate tissue represent their original content, we compared their levels with those in the fresh tissue (on dry weight basis) in samples obtained from 15 patients. We found that in FFPE tissue, the recoveries of Se, Fe, Cd and Zn were progressively decreased, 97±11% ( r=0.88), 82±22% ( r=0.86), 59±23% ( r=0.69) and 24±11% ( r=0.38), respectively. Thus, the use of correction factors, determined as k=0.16 for Se, k=0.20 for Fe, k=0.27 for Cd and k=0.67 for Zn, is required to estimate the retrospective levels of these elements in the parental non-processed fresh (wet) prostate tissue. The technique used in this study enables the analysis of archival FFPE prostate tissue for the concentrations of Fe, Zn, Se and Cd to study association between the levels of these metals and prostate disease.

Children Are Likely to Suffer Most from Our Fossil Fuel Addiction

BackgroundThe periods of fetal and child development arguably represent the stages of greatest vulnerability to the dual impacts of fossil fuel combustion: the multiple toxic effects of emitted pollutants (polycyclic aromatic hydrocarbons, particles, sulfur oxides, nitrogen oxides, metals) and the broad health impacts of global climate change attributable in large part to carbon dioxide released by fossil fuel burning.ObjectivesIn this commentary I highlight current scientific evidence indicating that the fetus and young child are at heightened risk of developmental impairment, asthma, and cancer from fossil fuel pollutants and from the predicted effects of climate disruption such as heat waves, flooding, infectious disease, malnutrition, and trauma. Increased risk during early development derives from the inherently greater biologic vulnerability of the developing fetus and child and from their long future lifetime, during which early insults can potentially manifest as adult as well as childhood disease. I cite recent reports concluding that reducing dependence on fossil fuel and promoting clean and sustainable energy is economically feasible.DiscussionAlthough much has been written separately about the toxicity of fossil fuel burning emissions and the effects of climate change on health, these two faces of the problem have not been viewed together with a focus on the developing fetus and child. Adolescence and old age are also periods of vulnerability, but the potential for both immediate and long-term adverse effects is greatest when exposure occurs prenatally or in the early years.ConclusionsConsideration of the full spectrum of health risks to children from fossil fuel combustion underscores the urgent need for environmental and energy policies to reduce fossil fuel dependence and maximize the health benefits to this susceptible population. We do not have to leave our children a double legacy of ill health and ecologic disaster.

Involvement of SREBPs in 2,3,7,8-tetrachlorodibenzo- p-dioxin-induced disruption of lipid metabolism in male guinea pig

2,3,7,8-Tetrachlorodibenzo- p-dioxin (TCDD) has multiple toxic effects causing a wasting syndrome characterized by a loss of body weight accompanied by a decrease in adipose tissue weight. To elucidate the mechanism behind this syndrome, we investigated the changes in lipid metabolism 7 and 21 days after a single intraperitoneal injection of TCDD at 1 μg/kg body weight to male guinea pigs. TCDD caused the symptoms of the syndrome, body weight loss with a decrease in adipose tissue weight, while it increased the levels of triacylglycerols, total cholesterols, and free fatty acids in plasma. On day 7, TCDD decreased the levels of CCAAT/enhancer binding protein (C/EBP) α, peroxisome proliferator activated receptor γ, and glucose transporter 4, adipogenesis-related factors, in adipose tissue, whereas the levels of retinoid X receptor α, C/EBPβ, C/EBPδ, and c-Myc remained unchanged. TCDD also reduced the levels of both p125 precursor and p68 active forms of sterol regulatory element binding protein (SREBP)-1 and -2, the lypogenesis-related factors, and downregulated their DNA binding activity in adipose tissue, while it raised the levels of their p68 active forms and increased their DNA binding activity in the liver. TCDD decreased mRNA and protein levels of acetyl-CoA carboxylase and HMG-CoA synthase in the liver and adipose tissue. Similar results were obtained on day 21. These results suggest that TCDD disrupts lipid metabolism through changes in the expression levels of the adipogenesis-related and lipogenesis-related proteins in the liver and adipose tissue, and SREBPs would be involved in the development of the wasting syndrome.

Heat shock proteins (Hsp 70) response is not systematic to cell stress: Case of the mycotoxin ochratoxin A

Ochratoxin A (OTA) is a mycotoxin routinely detected in improperly stored animal and human food supplies as well as in human sera worldwide. OTA has multiple toxic effects; however, the most prominent is nephrotoxicity. Thus, OTA is involved in the pathogenesis of human nephropathy in Balkan areas. In this study, we address the question of the appropriate functioning of the basal cellular defense mechanisms, after exposure to OTA, which, up to now, has not been investigated satisfactorily. In this context, we have monitored the effect of OTA on (i) the inhibition of cell viability, (ii) the oxidative damage using the GSH depletion, (iii) the inhibition of protein synthesis through the incorporation of [ 3H] Leucine and (iv) the induction of Hsp 70 gene expression as a parameter of cytotoxicity, oxidative damage and particularly as a protective and adaptative response. This study was conducted using the Human Hep G2 hepatocytes and monkey kidney Vero cells under exposure conditions ranging from non-cytotoxic to sub-lethal. Our results clearly showed that OTA inhibits cell proliferation, strongly reduces protein synthesis and induces the decrease of GSH in concentration-dependent manner in both Hep G2 and Vero cells. However, although cytotoxicity and oxidative damage (main inducers of Hsp expression) occur, no change was observed in Hsp 70 level under the multiple tested conditions. Inhibition of protein synthesis could not explain the absence of Hsp 70 response since concentrations, which did not influence protein synthesis, also failed to display the expected Hsp 70 response. Our data are consistent with recently published reports where considerable differences were noticed in the ability of relevant toxicants to induce Hsp. These results raised doubt about the universal character of Hsp induction which seems to be more complex than originally envisioned. It could be concluded that Hsp 70 induction is not systematic to cell stress.

Disruption of glucocorticoid action by environmental chemicals: Potential mechanisms and relevance

Glucocorticoids play an essential role in the regulation of key physiological processes, including immunomodulation, brain function, energy metabolism, electrolyte balance and blood pressure. Exposure to naturally occurring compounds or industrial chemicals that impair glucocorticoid action may contribute to the increasing incidence of cognitive deficits, immune disorders and metabolic diseases. Potentially, “glucocorticoid disruptors” can interfere with various steps of hormone action, e.g. hormone synthesis, binding to plasma proteins, delivery to target cells, pre-receptor regulation of the ratio of active versus inactive hormones, glucocorticoid receptor (GR) function, or export and degradation of glucocorticoids. Several recent studies indicate that such chemicals exist and that some of them can cause multiple toxic effects by interfering with different steps of hormone action. For example, increasing evidence suggests that organotins disturb glucocorticoid action by altering the function of factors that regulate the expression of 11β-hydroxysteroid dehydrogenase (11β-HSD) pre-receptor enzymes, by direct inhibition of 11β-HSD2-dependent inactivation of glucocorticoids, and by blocking GR activation. These observations emphasize on the complexity of the toxic effects caused by such compounds and on the need of suitable test systems to assess their effects on each relevant step.

Lithium- and Valproate-Induced Alterations in Circadian Locomotor Behavior in Drosophila

Lithium and valproate are commonly used mood stabilizers, but their action pathways are not clearly understood. They also suffer from multiple toxic effects that limit their utility. Elucidating their action mechanisms could lead to newer agents and better understanding of the etiopathogenesis of bipolar disorder. We have expanded the study of signaling mechanisms of lithium and valproate by using Drosophila circadian locomotor activity as a robust behavioral assay that is amenable to genetic manipulations. We demonstrate that lithium affects the circadian system of Drosophila similarly to what has been reported in the mammalian studies. We show that lithium and valproate share effects on the circadian locomotor activity of Drosophila: they lengthen the period of circadian rhythms and increase arrhythmicity. Valproate exerts these effects in a weaker fashion than does lithium. We also tested the circadian alterations in multiple mutant lines of Drosophila bearing defects in the GSK-3beta gene and other clock genes in response to lithium administration. We show that lithium partially rescues the shortening of circadian period when the GSK-3beta gene is overexpressed only in specific circadian pacemaker neurons, thus implicating GSK-3beta as a component in lithium's effect on the circadian oscillator. Moreover, lithium also lengthens the period in GSK-3beta heterozygous mutants and doubletime long mutants. These results establish a basis for using Drosophila genetics to investigate more fully lithium and valproate action mechanisms.

Small-for-Size Syndrome after Living-Donor Liver Transplantation Treated by “Portal Vein Wrapping” and Single Plasmapheresis

With the increasing use of living-donor liver grafts in adult patients, small-for-size syndrome became an important clinical problem (1). Its occurrence appears to depend on a number of recipient and graft factors. Potential pathogenic mechanisms include persistent portal hypertension and portal over-perfusion (2). At present, several techniques are being explored in an attempt to ameliorate the impact of small-for-size syndrome (3). A 47-year-old man, 196 cm tall and 78 kg in weight, suffering from end-stage hepatitis B cirrhosis, underwent living-donor liver transplantation (LDLTx) in our center. His own liver had a calculated volume of 1,639 mL as shown by native liver computer tomography (CT) and was replaced by the right hemiliver of his brother, which had a calculated native volume of 935 mL as shown by three-dimensional CT. A graft-recipient weight ratio of 0.8% was measured preoperatively. Intraoperatively, after an uneventful right hepatectomy of the donor according to the virtual intervention planning, the right graft was weighed at 719 g. To reduce the portal vein over-perfusion and to prevent a small-for-size syndrome in the recipient, a “portal vein-wrapping” with Tabotamb (regenerated oxidized cellulose mesh with local hemostatic ability) was performed intraoperatively. On the first postoperative day, the patient underwent a second laparotomy under the suspicion of hepatic artery thrombosis, which was not confirmed. Over the next 3 days, the patient developed liver dysfunction (elevation of total bilirubin from 4.4 to 31.2 mg/dL, increased prothrombin time [PT]) and increasing encephalopathy, although the other enzymes (ASAT, ALAT, LDH) showed a regular clinical course. A decision to implement plasmapheresis was made. The procedure was well tolerated by the patient, who remained hemodynamically stable throughout the treatment period. Serum levels of total bilirubin and PT as well as encephalopathy were significantly improved after the first session of plasmapheresis. Because of significant clinical improvement after the first session, plasmapheresis was not repeated. The patient left the intensive care unit on postoperative day 12. Liver volume was calculated as 2,137 mL on postoperative day 15. Bilirubin, ASAT, ALT, and coagulation profile returned to a normal range, and the patient was discharged on postoperative day 29 in good physical condition. Twelve months after transplantation, he returned to his everyday activities and is currently in good general condition. Severe hyperbilirubinemia is known to exert multiple toxic effects. Artificial liver-support devices attempt to bridge patients with fulminant hepatic failure until either a suitable liver allograft is obtained for transplantation or the patient’s own liver regenerates sufficiently to resume normal function (4). No great experience with liver-transplant patients is available as yet (5). The role of repeated sessions of plasmapheresis for primary allograft nonfunction after liver transplantation has been reported (6). As our case indicates, even a single plasmapheresis can be an effective treatment for small-for-size syndrome after LDLTx when combined with a surgical modulation of the portal vein inflow such as “portal vein wrapping.” Fuat Saner Georgios C. Sotiropoulos Arnold Radtke Massimo Malagó Christoph E. Broelsch Department of General Surgery and Transplantation University Hospital Essen Essen, Germany Stefan Herget-Rosenthal Department of Nephrology University Hospital Essen Essen, Germany

Preventive effect of Thea sinensis melanin against acetaminophen-induced hepatic injury in mice.

The preventive effect of Thea sinensis melanin (TSM) against overdoses of N-acetyl-p-aminophenol (NAPAP) was studied on ICR mice. Animals were given 400 mg/kg intraperitoneally (i.p.) of NAPAP, and TSM was injected i.p. in doses 10-40 mg/kg 2 h before intoxication. The protective effects were evidenced by a complete blockage of the NAPAP-induced elevation of plasma alanine aminotransferase (ALT) activity, decreased concentration of thiobarbituric acid reactive substances (TBARS) to the control level, and a partial prevention of reduced glutathione (GSH) depletion in the liver tissue. Preadministration of TSM also caused restoration of superoxide dismutase (SOD) activity and resumed content of coenzymes Q9 and Q10. TSM by itself, however, did not affect the hepatic functional parameters, including serum ALT, TBARS, GSH, SOD, or coenzymes Q in the liver. Administration of TSM caused a dose-dependent inhibition of N-nitrosodimethylamine demethylase activity with ED50 of 15.8 mg/kg. Activities of ethoxyresorufin O-dealkylase and pentoxyresorufin O-alkylase isozymes were changed insignificantly. The immune suppressive effect of NAPAP on the in vivo antibody-forming cell responses was demonstrated using ICR-sensitized mice with sheep red blood cells. The joint effect of TSM and NAPAP indicated the capability of TSM to recover immunity of the animals to the level of intact mice. Results obtained demonstrate that TSM preadministration can prevent the multiple toxic effects of NAPAP.

Relative effectiveness of calcium and magnesium in the alleviation of rhizotoxicity in wheat induced by copper, zinc, aluminum, sodium, and low pH

Root elongation in short-term experiments with wheat (Triticum aestivum L.) seedlings demonstrated that the following ions were rhizotoxic in the order Cu2+>Al3+≫ H+>Zn2+≫ Na+. Additions of Ca2+ and Mg2+ alleviated the toxicity, but the relative ameliorative effectiveness of Ca2+ and Mg2+ depended upon the toxicant. The effectiveness of Mg2+ relative to Ca2+ was 0.098 for Na+, 0.37 for H+, 1.0 for Al3+, 2.1 for Cu2+, and 170 for Zn2+. The mechanisms of inhibition are mainly unknown, but the mechanisms of alleviation are better understood. Mechanism I entails ameliorant-induced reduction of the negativity of the plasma membrane (PM) surface electrical potential (ψ0). The consequence is a reduced activity of the toxicant at the PM surface because of reduced electrostatic attraction. Ca2+ and Mg2+ are equally effective agents of Mechanism I alleviation but are less effective than H+ and more effective than Na+ for reasons described by electrostatic principles. Mechanism II alleviation is specific for Ca2+ and entails the restoration of Ca2+ at the PM surface if surface Ca2+ has been reduced by the toxicant to growth-limiting levels. This occurs more commonly in Na+ and H+ toxicities than in the others, though in no case is it the principal mechanism of alleviation. Mechanism III alleviation is the collective ameliorative effect of an ion beyond Mechanisms I and II. Differences between Ca2+ and Mg2+ in ameliorative effectiveness are mainly attributable to Mechanism III which, in the case of Zn2+, may entail an internal detoxification and, in the case of Na+, may entail the blockade of a Na+ uptake channel. This study demonstrates that appropriate nonlinear equations incorporating cell-surface ion activities enable the dissection of multiple toxic and ameliorative effects of the ions.

Molecular and cellular mechanisms of cadmium carcinogenesis.

Cadmium is a heavy metal, which is widely used in industry, affecting human health through occupational and environmental exposure. In mammals, it exerts multiple toxic effects and has been classified as a human carcinogen by the International Agency for Research on Cancer. Cadmium affects cell proliferation, differentiation, apoptosis and other cellular activities. Cd2+ does not catalyze Fenton-type reactions because it does not accept or donate electrons under physiological conditions, and it is only weakly genotoxic. Hence, indirect mechanisms are implicated in the carcinogenicity of cadmium. In this review multiple mechanisms are discussed, such as modulation of gene expression and signal transduction, interference with enzymes of the cellular antioxidant system and generation of reactive oxygen species (ROS), inhibition of DNA repair and DNA methylation, role in apoptosis and disruption of E-cadherin-mediated cell-cell adhesion. Cadmium affects both gene transcription and translation. The major mechanisms of gene induction by cadmium known so far are modulation of cellular signal transduction pathways by enhancement of protein phosphorylation and activation of transcription and translation factors. Cadmium interferes with antioxidant defense mechanisms and stimulates the production of reactive oxygen species, which may act as signaling molecules in the induction of gene expression and apoptosis. The inhibition of DNA repair processes by cadmium represents a mechanism by which cadmium enhances the genotoxicity of other agents and may contribute to the tumor initiation by this metal. The disruption of E-cadherin-mediated cell-cell adhesion by cadmium probably further stimulates the development of tumors. It becomes clear that there exist multiple mechanisms which contribute to the carcinogenicity of cadmium, although the relative weights of these contributions are difficult to estimate.

Effects of copper on wild and tolerant strains of the lichen photobiont Trebouxia erici (Chlorophyta) and possible tolerance mechanisms.

The influence of copper was assessed on wild and tolerant strains of the lichen photobiont Trebouxia erici and shown to have multiple toxic effects. Addition of 4 mM copper chloride into Trebouxia media reduced growth rates of the wild type to less than 1% of control levels. It also injured cell membranes, inhibited dehydrogenase activity, altered pigment composition, and reduced activity of photosystem II. Membrane damage was assessed through measuring electrical conductivity and potassium content, dehydrogenase activity by degree of conversion of 2,3,5-triphenyl tetrazolium chloride to red triphenylformazan, and functioning of PS II by chlorophyll a fluorescence. In respect to most parameters the tolerant strain was usually less affected by copper than the wild strain and, in some cases, not at all. We demonstrated by inductively coupled plasma atomic emission spectrometry and energy-dispersive X-ray microanalysis that enriched copper in the medium resulted in increased uptake by both wild and tolerant photobiont cells, but extracellular concentrations were significantly higher in the tolerant strain. This suggested that, at least in vitro, extracellular deposition was a mechanism of copper tolerance.

Mechanisms of the Deleterious Effects of Tamoxifen on Mitochondrial Respiration Rate and Phosphorylation Efficiency

Tamoxifen (TAM), the widely prescribed drug in the prevention and therapy of breast cancer, is a well-known modulator of estrogen receptor (ER) that also inhibits the proliferation of different cell types that lack the ER. However, the ER-independent action mechanisms of TAM and its side effects have not been yet clarified. Mitochondria are essential in supporting the energy-dependent regulation of cell functions. Changes in mitochondria result in bioenergetic deficits leading to the loss of vital functions to cell survival. Therefore, this study describes the effects of TAM on mitochondrial bioenergetics, contributing to a better understanding of the biochemical mechanisms underlying the multiple antiproliferative and toxic effects of this drug. TAM at concentrations above 20 nmol/mg protein, preincubated with isolated rat liver mitochondria at 25°C for 3 min, significantly depresses, in a dose-dependent manner, the phosphorylation efficiency of mitochondria as inferred from the decrease in the respiratory control and ADP/O ratios, the perturbations in mitochondrial transmembrane potential (ΔΨ), the fluctuations associated with mitochondrial energization, and the phosphorylative cycle induced by ADP. Furthermore, TAM at up to 40 nmol/mg protein stimulates the rate of state 4 respiration and at higher concentrations it strongly inhibits state 3 and uncouples the mitochondrial respiration. The stimulation of state 4 respiration parallels the decrease of ΔΨ as a consequence of proton permeability. The TAM-stimulatory action of ATPase is also observed in intact mitochondria, suggesting that TAM promotes extensive permeability to protons due to destructive effects in the structural integrity of the mitochondrial inner membrane. These multiple effects of TAM on mitochondrial bioenergetic functions, causing changes in the respiration, phosphorylation efficiency, and membrane structure, may explain the cell death induced by this drug in different cell types, its anticancer activity in ER-negative cells, and its side effects.

Molecular mechanism of lung hemorrhage induction by VRV-PL-VIIIa from Russell's viper ( Vipera russelli) venom

The basic phospholipase A 2 (VRV-PL-VIIIa) from Vipera russelli venom induces multiple toxic effects including neurotoxicity, myotoxicity, edema and hemorrhage. This phospholipase A 2 has been extensively characterized for its pharmacological properties except for hemorrhagic activity. In the present investigation, the lung hemorrhagic activity was assayed using lung dye diffusion method. The investigations to understand the mechanism of lung hemorrhage induction by VRV-PL-VIIIa was followed by chemical modification studies and also by interaction with an antihemorrhagic factor p-anisic acid (4-methoxy benzoic acid). In presence of 1:2 mol:mol PLA 2: anisic acid, the lung hemorrhagic and edema inducing activities were completely neutralized in experimental animals; however, catalytic and anticoagulant activities were not neutralized. Carbamylation of VRV-PL-VIIIa resulted in the loss of lung hemorrhage and edema inducing activities. In contrast, carbamylation of VRV-PL-VIIIa in the presence of anisic acid could not neutralize the lung hemorrhage and edema inducing activities. The anticoagulant and enzyme activities were only partially neutralized when carbamylated both in the presence and absence of anisic acid.

Markov chain models for multivariate repeated binary data analysis

Repeated categorical outcomes frequently occur in clinical trials. Muenz and Rubinstein (1985) presented Markov chain models to analyze binary repeated data in a breast cancer study. We extend their method to the setting when more than one repeated outcome variable is of interest. In a randomized clinical trial of breast cancer, we investigate the dependency of toxicities on predictor variables and the relationship among multiple toxic effects.

Interactions among Ca2+, Na+ and K+ in salinity toxicity: quantitative resolution of multiple toxic and ameliorative effects

Interactions among Ca2+, Na+ and K+ in salinity toxicity: quantitative resolution of multiple toxic and ameliorative effects

Interactions among Ca2+, Na+ and K+ in salinity toxicity: quantitative resolution of multiple toxic and ameliorative effects

Root elongation by wheat seedlings (Triticum aestivum L. cv. Scout 66) was not inhibited by NaCl or KCl up to 130 mM in culture solutions or by high Na+ (2 mg g−1 FW) or K+ (4 mg g−1 FW) in the root tissue, provided that [Ca2+]>2 mM in the rooting medium. At [NaCl], [KCl], or [mannitol] >250 mOs, root elongation was progressively inhibited, irrespective of high [Ca2+]. In contrast, shoot elongation was sensitive to any diminution of water potential, and Ca2+ alleviated the toxicity only weakly. At solute concentrations <250 mOs, the following interactions were observed. Ca2+ alleviated Na+ and K+ toxicity to roots by at least three separate mechanisms. K+ was more toxic to roots than Na+, but Na+ was more toxic to shoots. Low levels of K+ relieved Na+ toxicity, but low levels of Na+ enhanced K+ toxicity. Tissue concentrations of Na+ were reduced by Ca2+ and K+ in the rooting medium, and tissue concentrations of K+ were enhanced by Ca2+ and Na+. Several hypotheses relating to salinity toxicity can be evaluated, at least for wheat seedlings. The osmoticant hypothesis (salinity intoxication occurs because of diminished water potential) is true for shoots at all salinity levels, but is true for roots only at high salinity. The Ca2+-displacement hypothesis (Na+ is toxic because it displaces Ca2+ from the cell surface) is correct, but often of minor importance. The K+-depletion hypothesis (Na+ is toxic because it causes a loss of K+ from plant tissues) is false. The Cl−-toxicity hypothesis (the apparent toxicity of Na+ is induced by associated Cl−) is false. The results indicate that, apart from osmotic effects, high levels of Na+ in the rooting medium and in the tissues are not toxic unless Ca2+ is also deficient, a condition probably leading to inadequate compartmentation and excessive cytoplasmic accumulation. This study related growth to ion activities at plasma-membrane surfaces. These activities were computed by a Gouy-Chapman-Stern model then incorporated into non-linear growth models for growth versus toxicants and ameliorants.

Experimental model of cisplatin ototoxicity in chinchillas

Cisplatin is an important antineoplastic agent. Its ototoxicity has been well defined, both in human and animal studies. However, animal models of systemic cisplatin administration have been complicated by multiple toxic effects. We studied cisplatin ototoxicity in an animal model involving topical application of cisplatin to the round-window membrane. Adult chinchillas were anesthetized with ketamine and pentobarbital, and auditory function was tested with the use of auditory brain-stem responses to various stimuli (clicks and 8-and 16-kHz tone bursts). Each animal was used as its own control. The middle-ear cavity was exposed through the bulla. In the experimental ear, a 25-μl solution of 0.25 mg cisplatin/1.0 ml normal saline solution was applied to the round-window membrane. In the control ear, 25 μl normal saline solution was applied to the round-window membrane. Follow-up auditory brain-stem response testing was conducted 7 days after treatment. A significant increase in threshold in the experimental ears was seen on comparison with the control ears. This finding suggests that application of cisplatin to the round-window membrane is a useful animal model in which to study cisplatin ototoxicity. (Otolaryngol Head Neck Surg 1998;119:574-80.)

Hypersensitivity reactions to L-asparaginase do not impact on the remission duration of adults with acute lymphoblastic leukemia.

Among its multiple toxic effects, L-asparaginase induces allergic reactions that may reduce its biological effect. The impact of hypersensitivity reactions on the duration of leukemia-free survival (LFS) was assessed in adults with newly diagnosed acute lymphoblastic leukemia (ALL) receiving intensive multi-agent chemotherapy. In CALGB study 8811 (Blood 1995; 85: 2025-2037), 197 adults were scheduled to receive 14 doses of Escherichia coil L-asparaginase (6000 U/m2 SC) during 7 of the first 12 weeks of chemotherapy. No further L-asparaginase was given. Chemotherapy was given for 24 months. The median follow-up time has been 5.7 years. Of the 141 patients who remained on study after 12 weeks, 82 (58%) had received all 14 planned doses; 38 (27%) had 12-13 L-asparaginase doses documented in their treatment record; 21 (15%) patients had received < or =11 doses due to a variety of toxic effects. The mean number of doses received prior to experiencing any hypersensitivity reaction was seven (range 1-11). Seven patients had mild hypersensitivity reactions, but all seven eventually received 12-14 doses of E. coil L-asparaginase. Twenty-one other patients had severe hypersensitivity reactions that required discontinuation of E. coil L-asparaginase; 20 of these patients were switched to Erwinia L-asparaginase to complete their treatment. Ultimately, 12 of these 20 patients received 14 doses of L-asparaginase in total, and six received 12-13 doses. Thus, only three of the 21 patients who had severe hypersensitivity reactions received < or =11 total L-asparaginase doses. Other L-asparaginase-related complications included pancreatitis (15 patients), hypofibrinogenemia <100mg/dl (29 patients), and deep venous thrombosis or pulmonary embolism (eight patients); some of these patients had L-asparaginase discontinued after these complications. The estimates for LFS at 3 years were 55% (95% confidence interval, 44-65%) for the patients who received all 14 L-asparaginase doses (median LFS, 5.1 years), 47% (95% CI, 33-62%) for those who received 12-13 doses, and 48% (95% CI, 29-67%) for those who received < or =11 doses. There were no significant differences between these three groups in the length of LFS (P=0.68). LFS did not correlate with a history of severe hypersensitivity reaction (P=0.67). In general, E. coil L-asparaginase was well tolerated in these adult patients, and most patients received all of the planned therapy. Patients who had mild L-asparaginase hypersensitivity reactions and patients who switched to Erwinia L-asparaginase because of more severe allergic reactions did not have significantly shorter LFS than the remaining adults treated on this ALL protocol. The possibility that E. coli L-asparaginase is inactivated or destroyed in those individuals who have become hypersensitive to it becomes less important when allergic patients are secondarily treated with Erwinia L-asparaginase.

How aspartame prevents the toxicity of ochratoxin A.

The ubiquitous mycotoxin ochratoxin A (OTA) is found as a frequent contaminant of a large variety of food and feed and beverage such as beer, coffee and win. It is produced as a secondary metabolite of moulds from Aspergillus and Penicillium genera. Ochratoxin A has been shown experimentally to inhibit protein synthesis by competition with phenylalanine its structural analogue and also to enhance oxygen reactive radicals production. The combination of these basic mechanisms with the unusual long plasma half-life time (35 days in non-human primates and in humans), the metabolisation of OTA into still active derivatives and glutathione conjugate both potentially reactive with cellular macromolecules including DNA could explain the multiple toxic effects, cytotoxicity, teratogenicity, genotoxicity, mutagenicity and carcinogenicity. A relation was first recognised between exposure to OTA in the Balkan geographical area and Balkan Endemic Nephropathy (BEN) with a high incidence (nearly 50 times higher than normal) of urinary tract tumours. Exposure rates of OTA are measurable in blood of humans and animals and are established in several countries including Scandinavia, Germany, France, Italy, Canada, Japan and Northern Africa mainly Tunisia and Egypt. The impact of OTA exposure in non- endemic areas in the world is not known, the rates of exposure being not correlated with the disease records, especially in developed countries, due to lake of well- designed epidemiological studies, genetic polymorphism and maybe to dietary contents of radical scavengers and antioxidants. However the incidence and mortality rates of renal cancer are increasing in European countries and Northern Africa which could be a global resultant of human exposure to natural compounds in food such as mycotoxins and especially ochratoxin A. In addition to special care to prevent the growth of moulds and detoxification measures there was a need for the prevention of the OTA-induced toxic effects once the toxin is ingested. For this purpose several compound have been studied including some therapeutic agents such as piroxicam which cannot be proposed for a large scale use in humans for preventive purpose. Among other compounds, Aspartame, already used as sweetener has shown a real effectiveness in vivo confirmed largely in vitro. When rats exposed to OTA (289 micrograms/kg) by oral route every two days are given 25 mg/kg similarly for several weeks, all the toxic effects including genotoxicity are very efficiently prevented as shown for example by the disappearance of DNA- adducts in tissues excised from treated animals. Aspartame is also effective in washing out the toxin when given afterwards to animals intoxicated by the same oTA doses for several weeks. In vitro, provided that it is added in cell culture medium before OTA it prevent significantly the inhibition of protein synthesis and lipid peroxidation induced by the toxin. Obviously the molecular mechanism mediating the preventive effect of Aspartame is the delivery of phenylalanine by cleavage of the peptide but also the direct effect of the peptide on the bending capacity and transport of the toxin in vivo and in vitro. As a matter of fact when Aspartame is given to animals or added in culture medium the amount of peptide found unchanged (10-15%) may account for a preventive effect as entire peptide.

Monoclonal antibodies to VRV-PL-VIIIa, a basic multitoxic phospholipase A 2 from Vipera russelli venom

VRV-PL-VIIIa, the most basic phospholipase A 2 (PLA 2) from the venom of Vipera russelli, induces multiple toxic effects, including neurotoxicity, myotoxicity, edema and hemorrhage. Rabbit polyclonal antiserum was raised against VRV-PL-VIIIa. The antiserum cross-reacted in enzyme-linked immunosorbant assay (ELISA) with two other PLA 2 from the same venom, VRV-PL-V and VRV-PL-VI, and with ammodytoxin A, caudoxin and crotoxin. Twenty-two hybridoma cell lines secreting monoclonal antibodies against VRV-PL-VIIIa were isolated. The monoclonal antibodies exhibited apparent binding affinities in ELISA with VRV-PL-VIIIa ranging over two orders of magnitude. Most of the monoclonal antibodies cross-reacted moderately with VRV-PL-V and weakly with VRV-PL-VI. None of the antibodies cross-reacted with ammodytoxin, caudoxin or crotoxin. Reducing the disulfide bonds of VRV-PL-VIIIa lowered the ELISA signals of each monoclonal antibody to nonspecific levels, suggesting that all the antibodies recognize conformational epitopes. Four of the 22 antibodies neutralized the enzymatic activity of VRV-PL-VIIIa. Interestingly, two of the four exhibited the lowest affinities of the monoclonal antibody library for VRV-PL-VIIIa in ELISA, while the other two exhibited the highest. Each of the monoclonal antibodies was biotinylated and spatial binding relationships were evaluated by competition ELISA.