Showing papers in "Journal of Applied Toxicology in 2004"

Concentrations of parabens in human breast tumours

Abstract: Parabens are used as preservatives in many thousands of cosmetic, food and pharmaceutical products to which the human population is exposed. Although recent reports of the oestrogenic properties of parabens have challenged current concepts of their toxicity in these consumer products, the question remains as to whether any of the parabens can accumulate intact in the body from the long-term, low-dose levels to which humans are exposed. Initial studies reported here show that parabens can be extracted from human breast tissue and detected by thin-layer chromatography. More detailed studies enabled identification and measurement of mean concentrations of individual parabens in samples of 20 human breast tumours by high-pressure liquid chromatography followed by tandem mass spectrometry. The mean concentration of parabens in these 20 human breast tumours was found to be 20.6 +/- 4.2 ng x g(-1) tissue. Comparison of individual parabens showed that methylparaben was present at the highest level (with a mean value of 12.8 +/- 2.2 ng x g(-1) tissue) and represents 62% of the total paraben recovered in the extractions. These studies demonstrate that parabens can be found intact in the human breast and this should open the way technically for more detailed information to be obtained on body burdens of parabens and in particular whether body burdens are different in cancer from those in normal tissues.

Endocrine disrupters and human health: could oestrogenic chemicals in body care cosmetics adversely affect breast cancer incidence in women?

Abstract: In the decade that has elapsed since the suggestion that exposure of the foetal/developing male to environmental oestrogens could be the cause of subsequent reproductive and developmental effects in men, there has been little definitive research to provide conclusions to the hypothesis. Issues of exposure and low potency of environmental oestrogens may have reduced concerns. However, the hypothesis that chemicals applied in body care cosmetics (including moisturizers, creams, sprays or lotions applied to axilla or chest or breast areas) may be affecting breast cancer incidence in women presents a different case scenario, not least in the consideration of the exposure issues. The specific cosmetic type is not relevant but the chemical ingredients in the formulations and the application to the skin is important. The most common group of body care cosmetic formulation excipients, namely p-hydroxybenzoic acid esters or parabens, have been shown recently to be oestrogenic in vitro and in vivo and now have been detected in human breast tumour tissue, indicating absorption (route and causal associations have yet to be confirmed). The hypothesis for a link between oestrogenic ingredients in underarm and body care cosmetics and breast cancer is forwarded and reviewed here in terms of: data on exposure to body care cosmetics and parabens, including dermal absorption; paraben oestrogenicity; the role of oestrogen in breast cancer; detection of parabens in breast tumours; recent epidemiology studies of underarm cosmetics use and breast cancer; the toxicology database; the current regulatory status of parabens and regulatory toxicology data uncertainties. Notwithstanding the major public health issue of the causes of the rising incidence of breast cancer in women, this call for further research may provide the first evidence that environmental factors may be adversely affecting human health by endocrine disruption, because exposure to oestrogenic chemicals through application of body care products (unlike diffuse environmental chemical exposures) should be amenable to evaluation, quantification and control. The exposure issues are clear and the exposed population is large, and these factors should provide the necessary impetus to investigate this potential issue of public health.

Protective effects of caffeic acid phenethyl ester on doxorubicin-induced cardiotoxicity in rats

Abstract: The prevention of doxorubicin (DXR)-induced cardiotoxicity may be helpful to improve future DXR therapy The aim of this study was to investigate the cardio-protective effects of caffeic acid phenethyl ester (CAPE), an antioxidant agent, on DXR-induced cardiotoxicity Rats were divided into three groups and treated with saline, DXR and DXR + CAPE Rats were treated with CAPE (10 micromol x kg(-1) day(-1) ip) or saline starting 2 days before a single dose of DXR (20 mg x kg(-1) ip) Ten days later, haemodynamic measurements were performed and the hearts were excised for biochemical analyses and microscopic examination The heart rate and mean blood pressure were higher and the pulse pressure was lower in the DXR group than in the other two groups The administration of DXR alone resulted in higher myeloperoxidase activity, lipid peroxidation and protein carbonyl content than in the other groups The activities of superoxide dismutase and catalase were higher in DXR and DXR + CAPE groups than in the saline group Rats in the DXR + CAPE group had increased catalase activity in comparison with the DXR group and high glutathione peroxidase activity in comparison with the other two groups There was severe disruption of mitochondrial fi ne structure in the electron microscopy of the DXR group In contrast, myocardial microscopy appeared nearly normal in the DXR + CAPE group (as de fi ned at the electron microscopic level) In light of these in vivo haemodynamic, enzymatic and morphological results, we conclude that CAPE pretreatment significantly attenuated DXR-induced cardiac injury, possibly with its antioxidant effects

Role of caffeic acid phenethyl ester, an active component of propolis, against cisplatin-induced nephrotoxicity in rats.

Abstract: We have investigated the effect of caffeic acid phenethyl ester (CAPE) on cisplatin-induced nephrotoxicity in rats. Administration of a single dose of cisplatin resulted in the elevation of blood urea nitrogen and creatinine in serum, as well as nitric oxide in kidney tissue of rats. Cisplatin also caused reduction of catalase (P < 0.0001), superoxide dismutase (P = 0.149) and glutathrone peroxidase (P < 0.0001) activities in kidney tissue. Although cisplatin caused elevation in malondialdehyde levels and myeloperoxidase activities in kidney tissue, they were not statistically significant. Caffeic acid phenethyl ester was found to be protective against cisplatin-induced antioxidant enzyme reductions. Treatment with free-radical scavenger CAPE attenuated the increase in plasma blood urea nitrogen and kidney nitric oxide levels, and showed histopathological protection against cisplatin-induced acute renal failure. Extensive epithelial cell vacuolization, swelling, desquamation and necrosis were observed in the kidney of the cisplatin-treated rat. There were also larger tubular lumens in cisplatin-treated rats than those of the control and the CAPE groups. Caffeic acid phenethyl ester caused a marked reduction in the extent of tubular damage. It is concluded that administration of cisplatin imposes an oxidative stress to renal tissue and CAPE confers protection against the oxidative damage associated with cisplatin. This mechanism may be attributed to its free-oxygen-radical scavenging activity.

Detection of bioavailable heavy metals in EILATox-Oregon samples using whole-cell luminescent bacterial sensors in suspension or immobilized onto fibre-optic tips.

Abstract: At the EILATox-Oregon Workshop, nine luminescent whole-cell bacterial sensors were used for the determination of bioavailable metals in blind samples (17 synthetic and 3 environmental) A non-inducible luminescent control strain was used to determine sample matrix effects and bacterial toxicity Whole-cell bacterial sensors capable of determining arsenic, inorganic mercury and its organic derivatives, cadmium, lead or copper were used in suspensions and a bacterial sensor for the detection of inorganic mercury was immobilized onto fibre-optic tips using calcium alginate Bioavailable amounts of metals were estimated using calibration plots, that were constructed to determine the range of metals giving rise to a linear relationship between luminescence and the amount of metals present in the standard solutions EILATox-Oregon sample 5, which contained 74 mg l−1 of Hg, gave a significant response with both formats of the mercury sensor The bioavailable amounts of mercury according to the measurement of bacterial sensor in suspension and immobilized onto a fibre-optic tip were 76 and 93 mg l−1, respectively The bacterial sensor for arsenic and copper showed a response with sample 6 (58 mg l−1 of As) and sample 8 (400 mg l−1 of metham sodium), respectively This study showed that the bacterial sensors in suspension or immobilized onto optical fibres are capable of quantifying bioavailable metals from unknown samples The measurement protocol of bacterial sensors is simple and possible to perform in the field Moreover, the samples do not need any pretreatment before analysis Construction and characterization of the strain for the detection of bioavailable copper are described Copyright © 2004 John Wiley & Sons, Ltd

Significance of the detection of esters of p-hydroxybenzoic acid (parabens) in human breast tumours.

Abstract: This issue of Journal of Applied Toxicology publishes the paper Concentrations of Parabens in Human Breast Tumours by Darbre et al. (2004), which reports that esters of p-hydroxybenzoic acid (parabens) can be detected in samples of tissue from human breast tumours. Breast tumour samples were supplied from 20 patients, in collaboration with the Edinburgh Breast Unit Research Group, and analysed by high-pressure liquid chromatography and tandem mass spectrometry. The parabens are used as antimicrobial preservatives in underarm deodorants and antiperspirants and in a wide range of other consumer products. The parabens also have inherent oestrogenic and other hormone related activity (increased progesterone receptor gene expression). As oestrogen is a major aetiological factor in the growth and development of the majority of human breast cancers, it has been previously suggested by Darbre that parabens and other chemicals in underarm cosmetics may contribute to the rising incidence of breast cancer. The significance of the finding of parabens in tumour samples is discussed here in terms of 1) Darbre et al’s study design, 2) what can be inferred from this type of data (and what can not, such as the cause of these tumours), 3) the toxicology of these compounds and 4) the limitations of the existing toxicology database and the need to consider data that is appropriate to human exposures. Copyright © 2004 John Wiley & Sons, Ltd.

Effects of rosmarinic acid against aflatoxin B1 and ochratoxin-A-induced cell damage in a human hepatoma cell line (Hep G2)

Abstract: Recent findings have suggested that oxidative damage might contribute to the cytotoxicity and carcinogenicity of aflatoxin B1 (AFB1). The induction of oxidative stress also plays an important role in the toxicity of another mycotoxin: ochratoxin A (OTA). In this study, the protective effect of rosmarinic acid (Ros A) against AFB1 and OTA-induced cytotoxicity was investigated in a human hepatoma-derived cell line (Hep G2). Rosmarinic acid, a natural phenolic compound contained in many Lamiaceae herbs such as Perilla frutescens, sage, basil and mint, inhibits complement-dependent inflammatory processes and may have therapeutic potential. The ability of Ros A to reduce radical oxygen species (ROS) production, protein and DNA synthesis inhibition and apoptosis caused by the two mycotoxins was also investigated. Our experiments proved the significant cytoprotective effect of Ros A in vitro from OTA- and AFB1-induced cell damage. In particular, 24-h pretreatment with 50 µM Ros A inhibited the cytotoxicity of 10 µM AFB1 (by 45%) and 10 µM OTA (by 35%) in Hep G2 cells (P < 0.001). Moreover, Ros A dose dependently attenuated ROS production and DNA and protein synthesis inhibition induced by both of the toxins. Similarly, apoptosis cell death was prevented, as demonstrated by reduction of DNA fragmentation and inhibition of caspase-3 activation (P < 0.001). Copyright © 2004 John Wiley & Sons, Ltd.

Sidestream tobacco smoke as the main predictor of exposure to polycyclic aromatic hydrocarbons.

Abstract: Polycyclic aromatic hydrocarbons (PAHs) were measured in mainstream and sidestream tobacco smoke from 14 commercial brands of cigarettes purchased in Italy during 2001–2002. The PAHs detected in smoke and analysed with HPLC and a fluorimetric detector were: fluoranthene, pyrene, benzo[a]anthracene, chrysene, benzo[a]pyrene, benzo[b]fluoranthene, benzo[k]fluoranthene, benzo[g,h,i]perylene and dibenzo[a,h]anthracene. The PAH levels in mainstream smoke from different cigarette brands obtained using an official smoking machine varied by about threefold (from 118 to 374 ng per cigarette for total PAHs and from 23.5 to 100 ng per cigarette for carcinogenic PAHs). Total PAH levels in mainstream smoke were correlated with tar content (r = 0.615, P < 0.05, n = 14). Total PAH content in sidestream smoke, measured by collection of all the smoke produced by a lit cigarette in a glass chamber, was about tenfold higher compared with mainstream smoke. The PAH content in sidestream smoke was relatively uniform (2.3–3.9 and 0.49–1.21 µg per cigarette for total and carcinogenic PAHs, respectively) and was not correlated with tar content. These results indicate that cigarette manufacturing and filter characteristics influence the PAH composition of mainstream smoke, but have no effect on the PAH content in sidestream smoke, which is the main determinant of smokers' and non-smokers' exposure to PAHs in environmental tobacco smoke. Copyright © 2004 John Wiley & Sons, Ltd.

l‐Arginine ameliorates oxidative stress in alloxan‐induced experimental diabetes mellitus

Abstract: Oxidative stress occurs in diabetic patients and experimental models of diabetes. The ability of l-arginine to ameliorate the oxidative stress and metabolic changes after treatment with alloxan was investigated in rats. Adult male rats were injected intraperitoneally with 100 mg kg(-1) of alloxan to produce experimental oxidative stress characteristic of diabetes mellitus. Hyperglycaemia and hypercholesterolaemia were observed in serum after 7 days of alloxan treatment. This was associated with a depression of glutathione (GSH) concentration as well as superoxide dismutase (SOD) and catalase (CAT) activities in the liver and brain. In addition, the thiobarbituric acid-reactive substances (TBARS) were significantly elevated, indicating increased lipid peroxidation and oxidative stress in the same tissues. Administration of 100 mg kg(-1) l-arginine for 7 days either before or after alloxan injection significantly ameliorated the oxidative stress evidenced by a lower TBARS and a higher level of the endogenous GSH concentration and SOD and CAT activities than alloxan-treated rats. These effects were paralleled by marked protection and partial prophylaxis against alloxan-induced hyperglycaemia and cholesterolaemia. Thus, these results showed that exogenously administered l-arginine might improve the clinical manifestation of diabetes mellitus and decrease the oxidative stress in the liver and brain. In addition, the study supports the beneficial effect of l-arginine, which might be attributed to its direct, NO-dependent antioxidant capacity and/or NO-independent pathways.

Mutagenicity of textile dye products.

Abstract: Within an EU-funded research project, 281 textile dye products in use at 9 textile finishing companies from 8 European countries were assessed for potential mutagenic properties. Most of the dyes belonged to the so-called existing substances. Data sources considered were data published in the literature, unpublished industrial data provided by dye producing companies and laboratory testing. Data on mutagenicity are virtually absent for many of the dyes. Unpublished test results performed on behalf of the dye manufacturing industry proved to be an important data source, which is not accessible under usual circumstances. Four dye stuffs contained in seven dye products in use at the textile finishing companies were judged to be mutagenic based on published data from literature. Mutagenicity testing using Salmonella typhimurium, strains TA98 and TA100, revealed positive results for about 28% (15 out of 53) of the dye products investigated. Upon further testing with the mouse lymphoma assay (L5178Y/TK +/- ) 67% (6 out of 9) of Ames-positive dyes proved to be mutagenic in this mammalian cell test. All data sources combined led to an overall assessment as being mutagenic for 14 dye products out of 281. For 16 there is a suspicion of mutagenicity due to positive responses in one test. Still, 71 of the dye products are without any data on mutagenicity. This paper describes the data aggregation process, evaluation criteria, the overall assessment, and exemplifies controversial evaluations.

Glioma cell antioxidant capacity relative to reactive oxygen species produced by dopamine.

Abstract: The pathology of Parkinson's disease involves oxidative damage to dopaminergic neurons of the substantia nigra. Oxidation of the dopamine (DA) neurotransmitter itself may contribute to the generation of a reactive oxygen species (ROS) and subsequent neurodegeneration. Glia cells can either exacerbate injury or exert protective properties on local neurons in the brain. We investigate glial antioxidant enzyme systems relative to ROS generated during cytokine activation, monoamine oxidase (MAO) activity and autoxidation of DA in glioma cells. Rat C6 glioma cells stimulated with lipopolysaccharide Escherichia coli 0111:B4 and interferon gamma (LPS/IFN-g) produced high levels of nitric oxide (241 nmol mg(-1) protein 24 h(-1)) but not superoxide (O(-) (2)) or hydrogen peroxide (H(2)O(2)). Basal C6 cells exhibited a rapid and robust capacity to remove exogenous H(2)O(2) within minutes. Preincubation with sodium azide but not buthionine-[S, R]-sulfoximine attenuated this response, indicating catalase as the primary enzyme responsible for this effect. The glioma catalase reaction rate was slightly attenuated by exposure to LPS/IFN-g for 24 h. However, the reduction in catalase activity was not due to nitric oxide, because both the supernatant and sodium nitroprusside had no effect on isolated catalase enzyme activity. Hydrogen peroxide was produced only through substrate-driven MAO activity in prepared lysate. However, the quantity of H(2)O(2) produced per unit time (0.46 nmol mg(-1) protein min(-1)) was negligible compared with the enormous capacity for its removal by catalase (213.9 nmol mg(-1) protein min(-1)) (> or =462 x greater). Similarly, H(2)O(2) generated by DA autoxidation per unit time (0.28 nmol mg(-1) protein equiv. min(-1)), was rapidly dissolved by glioma cells at high capacity (> or =750 x greater). In conclusion, C6 cells produce nitric oxide under cytokine/endotoxin-stimulated conditions. Moreover, C6 cells exhibit a dynamic H(2)O(2) scavenging capacity, with ample facility to dispose of the peroxide generated by both MAO activity and spontaneous DA autoxidation.

Acetylcholine and choline effects on erythrocyte nitrite and nitrate levels.

Abstract: Acetylcholine has been detected in human blood. Acetylcholine receptors and acetylcholinesterase are present in erythrocyte membranes. We tested the acetylcholine and choline effects on nitric oxide metabolites (NOx), namely nitrites and nitrates, and observed if they are dependent on interactions with muscarinic receptors and acetylcholinesterase. Human erythrocyte suspensions were incubated with acetylcholine and choline in the absence or presence of 10 microM atropine or 10 microM velnacrine maleate. The nitrite and nitrate concentrations were determined by the Griess method. Acetylcholine or choline increased NOx control concentrations (P <0.001). The nitrite concentrations decreased in the presence of atropine or velnacrine maleate (P <0.03). The nitrate concentrations only decreased when velnacrine maleate was incubated with acetylcholine or choline (10 microM, P <0.03). These results demonstrated that acetylcholine and choline modulate nitric oxide metabolites on erythrocytes and this effect is mediated by interactions with erythrocyte membrane muscarinic receptors and membrane enzyme acetylcholinesterase. A hypothesis for the signal transduction mechanism has been discussed for acetylcholinesterase and muscarinic receptor (M1) participation.

Comparative sensitivity of Xenopus tropicalis and Xenopus laevis as test species for the FETAX model.

Abstract: The use of Xenopus tropicalis as an alternative test species for the Frog Embryo Teratogenesis Assay-Xenopus (FETAX) model was evaluated. Five test substances with varying developmental toxicity potential were evaluated using the traditional FETAX (X. laevis) and a modified assay to accommodate the use of X. tropicalis. Two separate definitive concentration-response tests were performed with ethanol, semicarbazide, copper, 6-aminonicotinamide (6-AN) and atrazine. In order to evaluate the impact of culture temperature on species sensitivity, tests with X. tropicalis were performed concurrently at 27 degrees C (optimum temperature) and 23 degrees C (traditional FETAX temperature). Tests with X. laevis were performed only at 23 degrees C (optimal for X. laevis). Regardless of culture temperature, tests with X. laevis and X. tropicalis indicated that each of the compounds possessed teratogenic potential: semicarbazide>6-AN>atrazine approximately copper>ethanol. Results from these studies indicated that these two species responded similarly to the test compounds. Xenopus tropicalis was somewhat less sensitive to 6-AN, semicarbizide and atrazine when tested at 27 degrees C than at 23 degrees C. Ethanol, copper and atrazine were reasonably equipotent in X. tropicalis and X. laevis in terms of teratogenic response (EC50 for malformation), whereas 6-AN and semicarbizide were less potent in X. tropicalis than in X. laevis. No substantial differences (order of magnitude) in potency were observed between X. laevis and X. tropicalis with any of the test materials evaluated. Malformation syndromes induced in both species were similar in X. tropicalis and X. laevis. These results suggested that X. tropicalis could be used effectively as a test organism for the FETAX model.

Amelioration of sulfur mustard skin injury following a topical treatment with a mixture of a steroid and a NSAID

Abstract: The ability to ameliorate sulfur mustard (HD)-induced oedema by treatment with anti-inflammatory drugs was reported previously after screening four steroids and four non-steroidal anti-inflammatory drugs (NSAIDs) using the mouse ear vesicant model. Following the screening study, one steroid and one NSAID (Adexone and Voltaren) were selected as the most effective, and a mixture of the two was chosen for the present more extensive research. The effect of the combined treatment on clinical, biochemical and histopathological parameters following HD insult was studied. Mice ears were exposed to 0.2 micro l of HD for 10 min to produce a moderate skin injury. Oedema development peaked ca. 48 h following exposure, as determined by weighing ear biopsies. Histological observations at that time exhibited damage to the epidermis and dermis. An increase in prostaglandin E (PGE) was measured in skin homogenates, starting 8 h following exposure and lasting at least up to 48 h post-exposure. A topical treatment using the above anti-inflammatory mixture significantly reduced inflammatory parameters when applied up to 4 h following exposure. These parameters included extent of oedema, levels of PGE, area of clinical damage and extent of cytotoxic injury (vesications and damaged epithelial cells). Thus, a combination of a steroid and NSAID was found to be effective in reducing the intensity of HD skin injury and possibly shortening the time to full recovery. The treatment, however, did not prevent completely the ensuing cytotoxic processes in the epithelial layer.

Protective effect of α-lipoic acid against chloroquine-induced hepatotoxicity in rats

Abstract: Oral administration of a-lipoic acid, a metavitamin, was investigated for its possible hepatoprotective effect in Wistar rats against chloroquine-induced toxicity. Rats were treated orally with alpha-lipoic acid (10, 30 and 100 mg x kg(-1) day(-1)) for 7 days before a single oral administration of chloroquine (970 mg x kg(-1) day(-1)) and alpha-lipoic acid treatment was continued for three more days. The increased level of serum enzymes (aspartate transaminase, alanine transaminase and alkaline phosphatase), bilirubin, lipids and plasma thiobarbituric acid-reactive substances (TBARS) and hydroperoxides observed in rats treated with chloroquine were very much reduced in rats treated with alpha-lipoic acid plus chloroquine. A significant decrease in plasma antioxidants such as reduced glutathione (GSH), vitamin C and vitamin E were observed in chloroquine-treated rats when compared with control rats. Administration of alpha-lipoic acid significantly improved the levels of plasma antioxidants GSH, vitamin C and vitamin E in chloroquine-treated rats. In the case of 100 mg x kg(-1) day(-1) the effect was highly significant compared with the other doses (10 and 30 mg x kg(-1) day(-1)). The results of the study revealed that alpha-lipoic acid could offer protection against chloroquine-induced hepatotoxicity. alpha-Lipoic acid had a better protective effect when compared with silymarin, a reference drug.

Delayed haematological complications of mustard gas.

Abstract: Haematopoiesis could be affected by mustard gas We randomly selected 318 chemical victims exposed to mustard gas and compared their cell blood counts and peripheral blood smears (PBS) with those of 377 healthy men, and also various haematological indices of 57 of these victims compared with previous data 5 years ago The average number of red blood cells and haemoglobin of victims compared with the controls was not significantly different, but they were increased compared with data from 5 years ago White blood cell counts, neutrophils and lymphocytes did not show any clinically meaningful difference compared with the control group but 20 cases with atypical lymphocytes in their PBS have been found Change in lymphocyte shape may be related to committed stem cell involvement Further studies on bone marrow cells and cell markers are needed to document this hypothesis The mild increase in erythroid cells and haemoglobin concentration may be due to chronic obstructive pulmonary disorder and other respiratory diseases in these patients

Response Characteristics of an Aquatic Biomonitor Used for Rapid Toxicity Detection

Abstract: The response characteristics of an aquatic biomonitor that detects toxicity by monitoring changes in bluegill (Lepomis macrochirus Rafinesque) ventilatory and movement patterns were evaluated in single chemical laboratory studies at concentrations near the 96-h LC(50) concentration and at the EILATox-Oregon Workshop in sequential tests of multiple unknown samples. Baseline data collected prior to exposure allows each fish to serve as its own control. When at least 70% of exposed fish exhibit ventilatory or movement parameters significantly different from baseline observations, a group alarm is declared. In the laboratory studies, the aquatic biomonitor responded to the majority of chemicals at the 96-h lc(50) within an hour or less, although substantially higher response times were found for malathion and pentachlorophenol. Workshop tests of single chemical concentrations presented as blind samples were consistent with the laboratory test results. There were no alarms under control conditions in any test. Although data are limited, the aquatic biomonitor appears to respond more rapidly to chemicals causing membrane irritation, narcosis or polar narcosis than to acetylcholinesterase inhibitors or oxidative phosphorylation uncouplers. All four monitored parameters (ventilatory rate, cough rate, ventilatory depth and movement) contributed to identification of first alarms at acutely toxic levels. Understanding these response patterns can be useful in data interpretation for biomonitor applications such as surface water monitoring for watershed protection, wastewater treatment plant effluent monitoring or source water monitoring for drinking water protection.

Qt prolongation in anaesthetized guinea-pigs: an experimental approach for preliminary screening of torsadogenicity of drugs and drug candidates

Abstract: Many non-cardiovascular drugs can prolong the QT interval of the electrocardiogram (ECG); this is an accessory property not necessary for their pharmacological action and generally linked to the block of the potassium HERG channels and delayed cardiac repolarization. The QT prolongation can lead to a dangerous tachyarrhythmia, called torsade de pointes, and potentially to fatal ventricular fibrillation. The experimental approaches, aimed at an early identification of this undesidered property, often require sophisticated and expensive equipment or the use of superior animal species (dog, primates) that cannot be employed easily for ethical and/or economic reasons. This work aimed to study drug-induced QT prolongation in anaesthetized guinea-pigs and to evaluate the reliability of such an experimental approach to obtain a satisfying predictive parameter of the torsadogenicity of drugs in humans. Seven drugs that were torsadogenic in humans (astemizole, cisapride, haloperidol, quinidine, sotalol, terfenadine and thioridazine) and two that were non-torsadogenic (chlorprotixene and diazepam) were administered i.v. to guinea-pigs under pentobarbital anaesthesia. The ECGs were recorded by four electrodes inserted in the subcutaneous layer of the limbs. Both RR and QT intervals were measured in Leads II and III and then the correct QT values were calculated by Bazett and Fridericia algorithms (QTcB and QTcF, respectively). All the drugs, with the exception of chlorprotixene and diazepam, produced a dose-dependent prolongation of the QT and RR intervals and a significant increase of QTcB and QTcF values. It can be concluded that this method represents a rapid and low-cost procedure to evaluate the cardiac safety pro fi le in the preliminary screening of a high number of drugs or drug candidates.

Cisplatin-induced acute renal failure is ameliorated by erdosteine in a dose-dependent manner.

Abstract: The aim of this study was to investigate the optimum dosage of erdosteine to ameliorate cisplatin-induced nephrotoxicity. Three different doses of erdosteine at 25, 50 and 75 mg kg−1 were studied in rats. Intraperitoneal administration of 7 mg kg−1 cisplatin led to acute renal failure, as indicated by kidney histology and increases in plasma creatinine and blood urea nitrogen (BUN) levels. At 5 days after cisplatin injection the BUN level was increased significantly from 15.1 ± 4.3 to 126.7 ± 152.6 mg dl−1 and plasma creatinine levels increased from 0.37 ± 0.005 to 1.68 ± 1.9 mg dl−1. When the rats were administered 50 and 75 mg kg−1 erdosteine 24 h before cisplatin injection that was continued until sacrifice (total of 6 days), the BUN and creatinine levels remained similar to control levels and the grade of histology was similar. Erdosteine at doses of 50 and 75 mg kg−1 ameliorates cisplatin-induced renal failure. The optimum dose of erdosteine may be 50 mg kg−1 in this study. Copyright © 2004 John Wiley & Sons, Ltd.

Water toxicity detection by a panel of stress-responsive luminescent bacteria

Abstract: A panel of Escherichia coli strains harbouring different stress-responsive promoters fused to a lux reporter system was used to assess the potential toxicity of 17 unknown model water samples. Using liquid cultures, nine out of 14 toxic samples were properly identified as toxic, whereas five were false negatives. All three non-toxic controls were identified correctly (no false positives). Two strains containing promoter-lux fusions were also tested when immobilized onto fibre-optic tips. One genotoxic sample and six toxic samples were correctly identified in this manner. The potential advantages and limitations in the use of genetically engineered bacteria as biosensors for water toxicity are discussed in view of these results.

Prevention of fumonisin-induced maternal and developmental toxicity in rats by certain plant extracts.

Abstract: In earlier work we have reported that garlic and cabbage extracts can protect laboratory animals from the toxic effects of different mycotoxins. Previous research demonstrated that fumonisin (FB) induced developmental effects in mice, rats and hamsters. The objectives of the present study were to utilize the pregnant rat as an in vivo model to compare the potential of garlic and cabbage seed extracts to prevent the developmental toxicity of FB and the effects of these extracts on sphingolipid metabolism in dam and foetus livers. Six treatment groups included a control group, a group fed on an FB-containing diet (150 mg kg−1 feed) and groups treated orally with garlic or cabbage extracts (5 mg kg−1 body wt.) with or without FB during gestation days 6–15. Evaluations of toxicity were performed on day 20. These include: maternal (mortality, body weight, feed intake and litter weight), developmental (embryonic resorption, foetal body weight, foetal soft-tissue anomalies and foetal skeletal examinations) and maternal and foetal sphingolipid metabolism. Fumonisin alone resulted in significant decreases in feed intake, body weight gain, litter weight, number of live foetuses and foetal body weight, whereas it increased significantly the number of resorbed foetuses and the number of skeletal malformations (30.4% for skull and 26.08% for sternebrae) and also increased the sphinganine/sphingosine (Sa/So) ratio in dam but not fetus livers. Garlic alone or plus FB was comparable to the control regarding all the tested parameters. On the other hand, cabbage seed extract alone or plus FB resulted in 10% maternal mortality and a decrease in maternal body weight and litter weight. It resulted in 4.65% skull malformations in foetuses but it was comparable to the control with regard to the other tested parameters. It could be concluded that both garlic and cabbage seed extracts have protective effects in pregnant rats. Moreover, garlic extract was found to have a greater protective effect than cabbage seed extract. Copyright © 2004 John Wiley & Sons, Ltd.

Liver microsomal biotransformation of nitro-aryl drugs: mechanism for potential oxidative stress induction.

Abstract: Toxic effects of several nitro-aryl drugs are attributed to the nitro-reduction that may be suffered in vivo, a reaction that may be catalysed by different reductases. One of these enzymes is NADPH–cytochrome P450 reductase, which belongs to the cytochrome P450 oxidative system mainly localized in the endoplasmic reticulum of the hepatic cell. This system is responsible for the biotransformation of oxidative lipophilic compounds, so that oxidative and reductive metabolic pathways of lipophilic nitro-aryl drugs can take place simultaneously. Because of the affinity of nitro-aryl drugs (xenobiotics) for the endoplasmic reticulum, we propose this subcellular organelle as a good biological system for investigating the toxicity induced by the biotransformation of these or another compounds. In this work we used rat liver microsomes to assess the oxidative stress induced by nitro-aryl drug biotransformation. Incubation of microsomes of rat liver with nifurtimox and nitrofurantoin in the presence of NADPH induced lipoperoxidation, UDP-glucuronyltransferase activation and an increase in the basal microsomal oxygen consumption. Nitro-aryl-1,4-dihydropyridines did not elicit these prooxidant effects; furthermore, they inhibited lipoperoxidation and oxygen consumption induced by Fe3+/ascorbate. Nifurtimox and nitrofurantoin modified the maximum absorption of cytochrome P450 oxidase and inhibited p-nitroanisole O-demethylation, an oxidative reaction catalysed by the cytochrome P450 system, signifying that oxidation may proceed in a similar way to that described for nitro-aryl-1,4-dihydropyridines. Thus the balance between lipophilic nitro-aryl drug oxidation and reduction may be involved in the potential oxidative stress induced by biotransformation. Copyright © 2004 John Wiley & Sons, Ltd.

Effects of ochratoxin a on oxidative damage in rat kidney: protective role of melatonin.

Abstract: Epidemiological studies indicate that ochratoxin A (OTA) may be involved in the pathogenesis of different forms of human nephropathies. Melatonin (MEL) displays antioxidant and free radical scavenger properties. In the present study, the effect of MEL on the oxidative stress induced by OTA administration in rats was investigated. Three groups of eight rats each were used: control, OTA (289 micro g kg(-1) day(-1)) and OTA + MEL (OTA, 289 micro g kg(-1) day(-1); MEL, 10 mg kg(-1) day(-1)), with treatment at two different time periods during the same day. After 4 weeks of treatment, the levels of lipid peroxidation (LPO) and the activities of catalase, superoxide dismutase (SOD) and glutathione peroxidase (GSHPx) were measured in homogenates of kidney. In OTA-treated rats, the levels of LPO and the activities of GSHPx and SOD were significantly decreased compared with controls. In OTA + MEL-treated rats, SOD, GSHPx and catalase activities and LPO levels were not changed significantly in comparison with controls.

Pyridoxine (vitamin B6) neurotoxicity: enhancement by protein-deficient diet.

Abstract: Large doses of pyridoxine cause injury to the primary sensory neurons in trigeminal and dorsal root ganglia of animals and patients subjected to megavitamin therapy. The increased hazard to subjects with reduced renal excretory function has been explored previously. In the present work, the neurotoxicity of pyridoxine for rats was found to be increased by dietary protein deficiency. A mere 3 or 7 days of pretreatment with either of two protein-deficient diets were sufficient to accelerate and intensify the clinical neurological signs and histological lesions from pyridoxine injections. These results are caused, at least in part, by loss of body weight, decreased protein binding in serum and decreased consumption of water and decreased volume of urine, which reduce the urinary losses of the toxicant. The vitamers related to pyridoxine (pyridoxal, pyridoxamine) and the coenzyme (pyridoxal 5-phosphate) did not cause clinical signs or lesions similar to those produced by pyridoxine even when injected in maximum tolerated doses. Neither a protein-deficient diet nor bilateral nephrectomy changed the results with the vitamers.

Pulmonary irritant potency of polyisocyanate aerosols in rats: comparative assessment of irritant threshold concentrations by bronchoalveolar lavage.

Abstract: The object of this study was to compare the relative acute pulmonary irritant potency of respirable aerosols of a variety of non-volatile polyisocyanates. The types of polyisocyanates examined included aliphatic homopolymers and mixed aliphatic–aromatic polyisocyanates consisting of the following monomers: HDI (hexamethylene 1,6-diisocyanate), IPDI (isophorone diisocyanate), MDI (methylene-diphenyl-4,4′-diisocyanate) and TDI (toluene diisocyanate). For reference purposes, the pulmonary irritant polyisocyanate aerosols were compared with monomeric IPDI, a semi-volatile respiratory tract (airway) irritant. In the substances tested, the concentration of free isocyanate moieties ranged from 11% to 38%. The relative potency to elicit pulmonary irritation was assessed by measurements of lung weights and total protein and lactate dehydrogenase (LDH) in the bronchoalveolar lavage fluid (BALF) following a single 6-h exposure of male rats. The time course of changes was analysed 3 h and 1, 3 and 7 days after exposure. When exposed to irritant concentrations of aerosol, BALF protein was maximal on post-exposure day 1 and returned to the level of the controls on post-exposure day 7. In contrast, rats exposed to sub-lethal concentrations of monomeric IPDI experienced a time-related increase in BALF protein. Based on this most sensitive endpoint, extrapolated no-observed-effect concentrations (NOECs) were in the range of 2–3 mg m−3 for most polyisocyanates examined. The NOECs from all the substances investigated were in the range 1–50 mg m−3. Thus, this methodology is adequate to rank the pulmonary irritant potency of polyisocyanate aerosols and to differentiate pulmonary from airway irritants. For pulmonary irritants the estimated acute NOECs were essentially similar to the no-observed-adverse effect concentrations (NOAECs) from long-term, repeated-exposure inhalation studies available for some of the polyisocyanates. A clear dependence of the NOAECs on the content of free isocyanate moieties was not observed. In summary, it is concluded that pulmonary irritation caused by polyisocyanate aerosols can be quantified readily in an acute rat bioassay by analysis of total protein in BALF. Moreover, this experimental evidence suggests that the NOECs of pulmonary irritants based on this endpoint are predictive of the NOAECs observed after subacute/subchronic inhalation exposure, suggesting that acute pulmonary irritation governs the outcome of repeated inhalation studies with such aerosols. However, for isocyanates where airway irritation predominates the pulmonary irritation, long(er)-term inhalation studies appear to be indispensable. The content of free NCO per se appears to be a poor predictor of the pulmonary irritant potency of polyisocyanate aerosols. Copyright © 2004 John Wiley & Sons, Ltd.

Mouse strain differences in eosinophilic airway inflammation caused by intratracheal instillation of mite allergen and diesel exhaust particles

Abstract: Response differences by different strains of mice towards house dust mites (Dermatophagoides farinae) or diesel exhaust particles (DEP) were investigated. Mouse strains BALB/c, ICR and C3H/He received 1 micro g of D. farinae or 1 microg of D. farinae + 50 microg of DEP intratracheally four times at 2-week intervals. Dermatophagoides farinae treatment caused the recruitment of eosinophils and lymphocytes. The order of magnitude of the eosinophilic airway inflammation was BALB/c < ICR < C3H/He mice. The protein levels of eotaxin and IL-5 in lung tissues correlated with the manifestations of eosinophilic airway inflammation by D. farinae administration. Diesel exhaust particles aggravated the manifestation of the eosinophilic inflammation through goblet cell proliferation in the airway and enhanced the local expression of eotaxin and IL-5 in all three strains of mice. The levels of eotaxin and IL-5 in lung tissues corresponded to the pathological changes caused by D. farinae + DEP. The increasing order of production levels of antigen-specific IgG1 by D. farinae or D. farinae + DEP was BALB/c < ICR < C3H/He mice. The significant adjuvant effect of DEP on IgG1 production was observed in the C3H/He mice (P < 0.05). These results suggest that the murine strain differences in the production of eosinophilic airway inflammation by D. farinae + DEP are related to differences in local expression of IL-5 and eotaxin. The enhancing effects of DEP may be mediated by a cytokine increase in the local expression. Antigen-specific IgG1 may be an important immunoglobulin in the pathogenesis of allergic asthma enhanced by DEP.

Succimer treatment and calcium supplementation reduce tissue lead in suckling rats.

Abstract: The effect of combined treatment with meso-2,3-dimercaptosuccinic acid (DMSA) and calcium supplementation in reducing lead absorption and enhancing lead elimination was evaluated in suckling rats under two experimental conditions: during ongoing oral lead exposure (lead acetate, 2 mg Pb kg−1 day−1, total dose 16 mg Pb kg−1) or after lead exposure (72 h after a 2-day lead exposure, total dose 12 mg Pb kg−1 s.c.). The artificial feeding method was used for calcium supplementation, with 6% Ca (as CaHPO4) suspension in cow's milk to increase the daily calcium intake about three times above control values. Artificial feeding lasted for 7 h a day over eight consecutive days. During this period DMSA was administered on 6 days twice a day (0.5 mmol kg−1 day−1 p.o.). At the end of the experiments, Pb, Ca and Zn in the carcass and Pb, Fe and Cu in the liver, kidneys and brain were analysed by atomic absorption spectrometry. Calcium supplementation during lead exposure reduced tissue lead but had no effect when applied after lead exposure, and DMSA administered either during or after lead exposure lowered the tissue lead. Combined treatment during ongoing lead exposure caused a greater reduction in tissue lead than either DMSA or calcium treatment alone. When administered after lead exposure, it had no advantage over DMSA treatment alone but did not impair its efficacy. Combined treatment had no influence on growth and did not seriously disturb essential element status. It is concluded that calcium supplementation could be applied during DMSA therapy, when indicated. Copyright © 2004 John Wiley & Sons, Ltd.

Cultured neuronal networks as environmental biosensors.

Abstract: Contamination of water by toxins, either intentionally or unintentionally, is a growing concern for both military and civilian agencies and thus there is a need for systems capable of monitoring a wide range of natural and industrial toxicants. The EILATox-Oregon Workshop held in September 2002 provided an opportunity to test the capabilities of a prototype neuronal network-based biosensor with unknown contaminants in water samples. The biosensor is a portable device capable of recording the action potential activity from a network of mammalian neurons grown on glass microelectrode arrays. Changes in the action potential firing rate across the network are monitored to determine exposure to toxicants. A series of three neuronal networks derived from mice was used to test seven unknown samples. Two of these unknowns later were revealed to be blanks, to which the neuronal networks did not respond. Of the five remaining unknowns, a significant change in network activity was detected for four of the compounds at concentrations below a lethal level for humans: mercuric chloride, sodium arsenite, phosdrin and chlordimeform. These compounds — two heavy metals, an organophosphate and an insecticide — demonstrate the breadth of detection possible with neuronal networks. The results generated at the workshop show the promise of the neuronal network biosensor as an environmental detector but there is still considerable effort needed to produce a device suitable for routine environmental threat monitoring. Copyright © 2004 John Wiley & Sons, Ltd.

Enzyme reactivator treatment in organophosphate exposure: clinical relevance of thiocholinesteratic activity of pralidoxime.

Abstract: Organophosphate compounds are responsible for a large number of accidental and/or suicidal exposures and have been used also for warfare and terrorism. The mechanism of toxicity is by inhibition of cholinesterase. Oximes are the only enzyme reactivators clinically available but clinical experience with oximes is disappointing. There is a gap between laboratory data and clinical impression concerning the efficacy of oxime compounds. Oximes are responsible for thiocholinesteratic activity, a spurious signal caused by interaction between pralidoxime and the thiocholine substrate used for photometric enzyme activity determinations. In a prospective, controlled, non-randomized study performed in anaesthetized miniature pigs, we quantified the extent of pralidoxime-induced cholinesteratic pseudo-activity ex vivo (human blood) and in vivo (minipig) in order to be able to correct values obtained by photometric methods. Plasma cholinesteratic activity using two substrates (acetylthiocholine and butyrylthiocholine) was determined in vitro and in vivo in the presence of pralidoxime. Pralidoxime reacts with the substrate (acetyl- and butyrylthiocholine) used for enzyme activity determinations, producing a spurious signal implying cholinesterase activity (even in the absence of plasma and thus of any enzyme). Cholinesterase activities determined photometrically after pralidoxime therapy can be erroneously high. Although in theory this could mislead clinicians into assuming an efficacious therapy, this is unlikely to occur in vivo under normal pralidoxime dosing conditions. To avoid any ambiguity it is recommended that blood be drawn for enzyme activity determinations prior to reactivator use and no less than 1 h after its administration.

Cytogenetic risk assessment of etoposide from mouse bone marrow.

Abstract: Increased clinical applications of the anticancer drug etoposide (a non-intercalative epipodophyllotoxin derivative) and the frequent induction of a second malignancy, particularly leukaemia, in post-etoposide-treated cancer survivors warrant detailed genotoxicity testing of etoposide. The genotoxicity test results available on etoposide are either primarily in in vitro test systems or in lower organisms after treatment with unusually high doses, or after chronic exposures, having little extrapolative value to humans. Therefore, a cytogenetic risk assessment study on etoposide in mouse in vivo was undertaken after a low dose (in accordance with the human therapeutic dose) single exposure. The cytogenetic toxicity of etoposide was assessed from bone marrow of mouse at three separate endpoints: chromosomal aberration and mitotic index studies at 24 h post-treatment and the micronucleus test (MNT) at 30 h post-treatment. The flame drying technique using colchicine, hypotonic sodium citrate, methanol-glacial acetic acid and Giemsa was followed for the preparation of slides for the metaphase chromosomal aberration and mitotic index studies and a simple technique was followed for the MNT. Although induction of chromosomal aberrations, excluding gaps, per 100 metaphases by 10 and 15 mg kg(-1) etoposide was not significant statistically, 20 mg kg(-1) of etoposide induced a significantly higher number of chromosomal aberrations in female (P < or = 0.01) and male (P < or = 0.05) mice. There was no significant change in the induced percentages of dividing cells by any of the doses of etoposide tested. The micronucleus induction also was not significant statistically with the lowest dose but it was significant in female (P