Experimental Toxicology Research Articles

Hormesis and precaution: the twain shall meet

Regulatory focus on quantifying risk of disease or death from exposure to hazardous substances via monotonic dose-response models has downplayed or even rejected potential benefits to human health from exposures to low (sub-threshold) doses, and thus represented by either U-shaped or J-shaped models. On the other hand, most environmental health policy hypothesizes, without firm evidence, that cancer risk is proportional to exposure at low doses of current ambient exposures. An acceptable exposure is determined by either setting a somewhat arbitrary ;acceptable' level of risk, such as one in a million excess individual lifetime cancer risk or, in the case of several types of animal toxicological test results, applying multiplicative safety factors to a specific concentration, generally derived from a benchmark dose or NOAEL. This seemingly precautionary approach is questionable in light of much experimental evidence indicating protective effects of exposure at low doses - U-shaped or J-shaped models. We demonstrate that incorporating the possibility of hormesis into regulatory decision-making is precautionary, while use of default results in policy conflicts with precaution.

Early nifurtimox-induced biochemical and ultrastructural alterations in rat heart

Nifurtimox (Nfx) and Benznidazole (Bz) are being used for the treatment of the acute phase of Chagas' disease. Recently, they were also considered for use in the indeterminate phase. Both the nitroheterocyclic drugs have serious toxic side effects. The mechanism of Nfx toxicity is associated with the formation of reactive oxygen species (ROS) generated during nitroreduction. Potential effects on cardiac function have not been established yet, despite the well-known cardiopathy often produced by the disease itself. We describe experiments testing some acute effects of Nfx on the male Sprague Dawley rat heart. Nifurtimox was present in the heart at 1, 3 and 6 h after intragastric (i.g) treatment. In vitro studies on Nfx microsomal and cytosolic nitroreductase activities showed that only the microsomal fraction had the ability to nitroreduce it. Cytochrome P450 and cytochrome P450 reductase would be involved in the process as suggested by their response to specific inhibitors. Nifurtimox increased the cardiac protein carbonyl content at 1 and 3 h and decreased the protein sulfhydryl content at 3 h. In addition, 24 h after treatment ultrastructural alterations such as marked cytoplasmic vacuolization, separation and loss of myofibrils and mitochondrial swelling were observed. Results suggest that Nfx administration might aggravate pre-existing adverse cardiac conditions. Human & Experimental Toxicology (2007) 26, 781 -788.

Functional alterations of rabbit erythrocytes induced by Loxosceles gaucho venom

Loxoscelism is the syndrome caused by the brown spider Loxosceles gaucho bite in humans. Its effect on erythrocytes has been studied in humans, rabbits, pigs and guinea pigs. In this study, the damage that the L gaucho spider venom causes to the structure and function of erythrocytes in vivo was investigated in rabbits. Before and after the rabbits were envenomed, membrane proteins were studied through polyacrylamide gel electrophoresis and membrane function was ascertained using the osmotic fragility test, together with the highly sensitive technique of ektacytometry. Six New Zealand rabbits were inoculated by intradermal injection into the dorsal region (10 microg of venom/kg of body weight in 0.2 mL of saline). Blood was collected at 24, 48, 72 and 120 h after inoculation. The membrane protein study did not reveal any alteration in their relative band concentrations, but the osmotic fragility test showed increased hemolysis in slightly hypotonic sodium chloride solutions (at 0.6 and 0.55%). In addition, the ektacytometer study revealed greater deformability to increasing shear stress on the order of 3-30 Pascals when compared with controls, showing that the L gaucho venom does in fact alter red cell function.

Comparative analysis of gene expression between renal cortex and papilla in nedaplatin-induced nephrotoxicity in rats

To elucidate the mechanism of nephrotoxicity caused by anti-neoplastic platinum complex, nedaplatin (NDP), treatment with a particular focus on the renal papillary toxicity, we analysed the gene expression profiles of two renal regions, the cortex (RC) and the papilla (RP) in rat kidneys. Male Wistar rats received a single administration of 10 mg/kg intravenous NDP or vehicle alone (5% xylitol solution) and were sacrificed six days later. The kidneys were dissected into the RC and RP and used for histopathological and microarray analyses. Histopathologically, NDP caused characteristic renal lesions, such as necrosis, single cell necrosis (with TUNEL TdT-mediated dUTP-biotin nick end labelling-positive) and regeneration/hyperplasia of the epithelial cells in both renal regions. Global gene expression analysis revealed that several genes involved in various functional categories were commonly deregulated in both renal regions, such as apoptosis, cell cycle regulation, DNA metabolism, cell migration/adhesion and cytoskeleton organization or genes induced as a perturbation of oxidative status and calcium homeostasis. Comparative analysis of gene expression between RC and RP revealed that genes encoding several subtypes of cytokeratins were identified as being specifically overexpressed in RP by the NDP treatment. Differential expression patterns of these selected genes observed by microarray analysis were further confirmed by quantitative real time RT-PCR and immunohistochemistry, which demonstrated increased expression of cytokeratins (CKs) 14 and 19 at the epithelium covering RP and/or collecting duct epithelium. Overall, the results contribute to understanding the renal molecular events of NDP-induced nephrotoxicity including novel potential biomarker genes encoding CKs 14 and 19 that may serve as indicators of renal papillary toxicity.

Arsenic neurotoxicity — A review

Arsenic (As) is one of the oldest poisons known to men. Its applications throughout history are wide and varied: murder, make-up, paint and even as a pesticide. Chronic As toxicity is a global environmental health problem, affecting millions of people in the USA and Germany to Bangladesh and Taiwan. Worldwide, As is released into the environment by smelting of various metals, combustion of fossil fuels, as herbicides and fungicides in agricultural products. The drinking water in many countries, which is tapped from natural geological resources, is also contaminated as a result of the high level of As in groundwater. The environmental fate of As is contamination of surface and groundwater with a contaminant level higher than 10 particle per billion (ppb) as set by World Health Organization (WHO). Arsenic exists in both organic and inorganic species and either form can also exist in a trivalent or pentavalent oxidation state. Long-term health effects of exposure to these As metabolites are severe and highly variable: skin and lung cancer, neurological effects, hypertension and cardiovascular diseases. Neurological effects of As may develop within a few hours after ingestion, but usually are seen in 2-8 weeks after exposure. It is usually a symmetrical sensorimotor neuropathy, often resembling the Guillain-Barré syndrome. The predominant clinical features of neuropathy are paresthesias, numbness and pain, particularly in the soles of the feet. Electrophysiological studies performed on patients with As neuropathy have revealed a reduced nerve conduction velocity, typical of those seen in axonal degeneration. Most of the adverse effects of As, are caused by inactivated enzymes in the cellular energy pathway, whereby As reacts with the thiol groups of proteins and enzymes and inhibits their catalytic activity. Furthermore, As-induced neurotoxicity, like many other neurodegenerative diseases, causes changes in cytoskeletal protein composition and hyperphosphorylation. These changes may lead to disorganization of the cytoskeletal framework, which is a potential mechanism of As-induced neurotoxicity.

Effects of chlorinated biphenyls and metabolites on human uterine myocyte proliferation

Uterine myometrial cells are responsive to sex steroids, which could make them susceptible to actions of endocrine disrupting environmental contaminants such as some PCBs. The aim of this investigation was to identify possible effects of some chlorinated biphenyls (CBs) and their metabolites on myometrial cell proliferation. Myometrial cells obtained from women in both phases of the menstrual cycle and from pregnant women were grown in vitro and exposed to CB 101, CB 118, 3' -MeSO2-CB 101, 4'-MeSO2-CB 101, 4-OH-CB 107, 17 beta-estradiol, progesterone, ethinylestradiol or levonorgestrel. The proliferative activity was studied by a BrdU assay. Myometrial cell cultures originating from pregnant women exhibited decreased proliferation in response to 3'-MeSO2-CB 101, 4'-MeSO2-CB 101 and 4-OH-CB 107. Estradiol, a combination of 1 nM 17beta-estradiol and 10 nM progesterone, ethinylestradiol and levonorgestrel also reduced the proliferation of the myometrial cells, regardless of whether the cells were collected from either of the menstrual cycle phases or from pregnant women. To our knowledge this study is the first to demonstrate that some CBs affect the proliferative activity of human uterine myocytes.

Determination of AChE levels and genotoxic effects in farmers occupationally exposed to pesticides

Pesticides can cause cytogenetic effects and lower the acetyl cholinesterase (AChE) levels in farmers exposed to pesticides. In this study, 210 farmers exposed to pesticides and 160 non-exposed individuals were enrolled for determining the genotoxicity and AChE levels. The AChE levels were determined in plasma and RBC lysate from blood samples collected from farmers and control subjects. AChE (true and pseudo) estimation done by the colorimetric method revealed that there was a progressive fall in both the RBC and plasma AChE levels in exposed individuals compared to unexposed individuals, which correlated with the severity of exposure (253.5 versus 311.1 and 142.3 versus 152.1; P < 0.001). Cytogenetic studies showed an increase in DNA damage and higher chromosomal aberrations (CAs) in exposed farmers compared to the control subjects (26.13 versus 07.61 and 21.37 versus 1.52; P < 0.001). When comparing the AChE levels with DNA damage and structural CA frequencies, there was a negative linear correlation. Therefore based on these findings, it is concluded that genotoxic biomarkers like CA frequencies, DNA damage data along with AChE levels are important parameters for determining farmer's health who are exposed to pesticides in any situation.

Alteration of estrogen-regulated gene expression in human cells induced by the agricultural and horticultural herbicide glyphosate

Gene expression is altered in mammalian cells (MCF-7 cells), by exposure to a variety of chemicals that mimic steroid hormones or interact with endocrine receptors or their co-factors. Among those populations chronically exposed to these endocrine disruptive chemicals are persons, and their families, who are employed in agriculture or horticulture, or who use agricultural/horticultural chemicals. Among the chemicals most commonly used, both commercially and in the home, is the herbicide glyphosate. Although glyphosate is commonly considered to be relatively non-toxic, we utilized in vitro DNA microarray analysis of this chemical to evaluate its capacity to alter the expression of a variety of genes in human cells. We selected a group of genes, determined by DNA microarray analysis to be dysregulated, and used quantitative real-time PCR to corroborate their altered states of expression. We discussed the reported function of those genes, with emphasis on altered physiological states that are capable of initiating adverse health effects that might be anticipated if gene expression were significantly altered in either adults or embryos exposed in utero.

Benzene's toxicity: a consolidated short review of human and animal studies by HA Khan

Khan's review is a brief summary of the complex field of study revolving around bone marrow toxicity and leukemogenesis observed in people chronically exposed to benzene. These comments are intended to demonstrate the use of the Kahn review as a launching pad for an in-depth analysis of the several related areas that must be fully explored to understand benzene-related diseases. The accumulated evidence demonstrates that benzene-induced bone marrow damage results from the production of hematotoxins that are metabolic products of benzene metabolism. The metabolism of benzene is described with respect to the formation benzene metabolites with emphasis on phenol and hydroquinone, which are the major metabolites, the significance of the formation of glutathione conjugates, the activity of NAD(P)H:quinone oxidoreductase (NQO1), and the ring opening products. Results are shown suggesting that oxidative stress induced by benzene metabolites is likely to be a significant factor in damaging DNA in bone marrow cells. Although a variety of effects on bone marrow can be demonstrated it is not yet clear which metabolites are most important in either benzene-induced aplastic anemia or leukemia. Benzene metabolism alone is insufficient to fully describe benzene toxicity. The impact of benzene metabolites on bone marrow cells must be fully explored to determine how benzene exposure can result in decreased viability or genetic toxicity to cells in the bone marrow.

Renal and hepatic injury with elevated cardiac enzymes in Amanita phalloides poisoning: a case report

Amatoxins are one of the most potent toxins that cause hepatic and renal failure. However, this is the first report demonstrating an elevation of cardiac enzymes in a patient with Amanita phalloides poisoning. A 56-year-old male was admitted to the emergency department (ED) 42 h after an unknown type of mushroom ingestion. Hepatic, renal function tests, amylase and cardiac enzymes (troponin I, creatine kinase (CK), CK-MB isoenzyme and myoglobin) were found elevated in his blood chemistry. The electrocardiogram disclosed sinus tachycardia. Aggressive treatment with fluids, activated charcoal, penicillin G and silibinin were started. The patient was sent to hemodialysis because of anuria. During follow-up, biochemical parameters and clinical findings improved. The patient was discharged from the hospital following the arrangement of hemodialysis schedule because of the chronic renal failure. False elevations of cardiac markers may confuse the clinicians in differential diagnosis of myocardial infarction in ED. In our patient, amatoxins that have bound the actin filaments within myocardiocytes or renal cells and/or its effects as circulating anti-troponin antibodies might result in elevation of cardiac markers. Elevated cardiac enzyme levels without any acute coronary syndrome are probable in mushroom poisoning cases involving amatoxin ingestion.

Pattern of acute poisoning in Tehran-Iran in 2003

To characterize the poisoning cases admitted to the Loghman-Hakim Hospital Poison Center (a teaching reference hospital of poisoning) in Tehran, Iran. All admitted acutely poisoned patients from January to December 2003 were evaluated retrospectively. Information of socio-demographic characteristics, agents and cause of poisoning, and the mortality rate were collected from medical records of the hospital. During this period, 24 179 cases were referred to the emergency department that 10 206 of them were admitted. Of the admitted cases, 51% were male and 49% female. The majority (38%) of cases were in the age range of 21-30 years. Most (79%) of poisonings were intentional and 21% were unintentional. The most important agents of acute poisoning were drugs (69.13%) especially sedative-hypnotics followed by opioids (12.34%) and pesticides especially organophosphates (OPs) (6.21%). The mortality rate was 1.3% (318 patients). Death was mostly occurred by opioids (41.54%), followed by drugs (28%) and pesticides especially OPs (12%). The prevention and treatment of poisoning due to opioids, pesticides specially OPs and sedative-hypnotics drugs should merit high priority in the health care of the indigenous population of Tehran.

Improvement by N-acetylcysteine of acute respiratory distress syndrome through increasing intracellular glutathione, and extracellular thiol molecules and anti-oxidant power: evidence for underlying toxicological mechanisms

In acute respiratory distress syndrome (ARDS), there is extensive overproduction of free radicals to the extent that endogenous anti-oxidants are overwhelmed, permitting oxidative cell damage. The present study examined the benefit of the anti-oxidant compound N-acetylcysteine (NAC) in the management of ARDS by measuring patient's intracellular glutathione (inside red blood cells) and extracellular (plasma) anti-oxidant defense biomarkers and outcome. Twenty-seven ARDS patients were recruited from the intensive care unit of a teaching Hospital and randomly divided into two groups. Both groups were managed similarly by regular treatments but 17 patients received NAC 150 mg/kg at the first day that followed by 50 mg/kg/day for three days and 10 patients did not receive NAC. Treatment by NAC increased extracellular total anti-oxidant power and total thiol molecules and also improved intracellular glutathione and the outcome of the patients. In conclusion, patients with ARDS are in a deficient oxidant-anti-oxidant balance that can get a significant benefit if supplemented with NAC.

Aluminum exposure decreases dopamine D1 and D2 receptor expression in mouse brain

Aluminum (Al) has been identified as a potential contributing factor in the etiology of several neurodegenerative disorders, but data regarding specific effects on neurotransduction, especially on dopaminergic neurotransduction, are lacking. The objective of this study was to determine the extent of expressional alterations in dopamine receptors (DRs) in two dopaminergic subtypes, D1 and D2, in low and high dose Al-treated mice. After administration of Al (four intraperitoneal injections of 30 or 60 mg/kg AlCl3.6H2O at 2 h intervals), expression of the dopamine D1-like and D2-like receptors (DRD1, DRD2) was examined in the cortex and striatum of mouse brain at bregma levels of 1.10, -0.10 and -1.34 mm. In the cortex, Al treatment decreased densities of DRD1 and DRD2 in a dose-dependent manner at all three bregma levels, especially in the high-dose Al group. Similarly, DRD1 and DRD2 expression in the striatum also exhibited dose dependency and statistically significant decreases were seen in the high-dose group, except in the striatum at bregma level - 1.34. These findings suggest that DR in the caudal striatum is more resistant to the effects of Al exposure than DR in the cortex or rostral striatum. In addition, our results suggest that disturbance of dopaminergic neurotransmission mediated by DRD1 and/or DRD2 may be involved in the pathogenesis of Al neurotoxicity.

Short Review: Benzene's toxicity: a consolidated short review of human and animal studies

A large population of humans is exposed to benzene from various occupational and environmental sources. Benzene is an established human and animal carcinogen. Exposure to benzene has been associated with leukaemia in humans and several types of malignancies in animals. The exact mechanism of benzene-induced toxicity is poorly understood. It is believed that benzene exerts its adverse effects by metabolic activation to toxic metabolites. Certain benzene metabolites are genotoxic and mutagenic. This consolidated short-review is composed of human and animal studies to summarize the adverse effects of benzene with special reference to molecular mechanisms involved in benzene-induced toxicity. Human &amp; Experimental Toxicology (2007) 26, 677— 685

Behavioral effects induced by subchronic exposure to Pb and their reversion are concentration and gender dependent

Lead (Pb) seems to be involved in the etiology of psychological pathologies. This study investigated the effects of subchronic Pb exposure from weaning to adulthood on anxiety, depression and aggressiveness in male and female Swiss mice. Moreover, the reversibility of the effects was evaluated retesting the animals 30 days after the end of exposure. Swiss male and female mice (21 days) were exposed to 0, 50, 100 or 500 ppm of Pb, as Pb acetate, in drinking water for 70 days and were submitted to the forced swimming, tail suspension, elevated plus-maze or intruder-resident tests. Pb exposure to 50 and 500 ppm induced anti-depressant-like effect in both males and females, whereas exposure to 500 ppm induced anxiogenic effect only in males. Interruption of exposure was able to reverse the behavioral alterations in females, but not in males exposed to the highest concentration (500 ppm). Our results suggest that behavioral effects induced by subchronic exposure to Pb from weaning to adulthood and their reversion are concentration and gender dependent.

Effects of amphetamine and cocaine on the development of acute experimental allergic encephalomyelitis in Lewis rats

The present experiment deals with the effects of amphetamine and cocaine on the development and course of experimental allergic encephalomyelitis (EAE) induced in Lewis rats. Rats were immunized at the age of eight weeks with purified myelin basic protein isolated from guinea pig brain in complete Freund's adjuvant. Drug administration and recording of EAE clinical signs was performed daily since day 1 post-immunization (PI). On day 14 and 28 PI, six rats per group were bled and sacrificed. Spinal cord was examined histologically for EAE lesions. In vivo administration of 0.5 and 1 mg/Kg of amphetamine or cocaine resulted in a dose-related enhancement of neurological and histological signs of acute EAE in comparison with control rats. Both drugs caused a reduction of latent period together with a delayed regression of neurological signs along with an increase in inflammation in the central nervous system in comparison with placebo. Human & Experimental Toxicology (2007) 26, 637-643.

Mitochondrial oxidative stress in female and male rat brain after ex vivo carbon monoxide treatment

Carbon monoxide (CO) is the most common cause of fatal poisoning all over the world. At the cellular level, a combination of tissue hypoxia and direct cellular damage underlie the pathophysiology of CO toxicity. The purpose of this study was to determine the effect of CO treatment on oxidative stress parameters in mitochondria isolated from male and female rat brains. Mitochondria prepared from frontal cortex, hippocampus and corpus striatum were treated with 0.1% CO at 37 degrees C for 30 minutes; control samples were not exposed to CO. Cytochrome c oxidase activity (COX), lipid peroxidation (thiobarbituric acid reactive species = TBARS), protein oxidation (protein carbonyls) and glutathione (GSH) levels were measured in CO treated and control samples. Our results confirmed previous studies reporting the inhibition of cytochrome c oxidase activity by CO in rat brain. Additionally, protein carbonyl levels in the hippocampus and striatum significantly increased after CO treatment in male rats. While CO treatment caused a significant decrease in GSH levels in the cortex and striatum in male rats, reduced GSH levels were observed in the cortex and hippocampus in female rats following CO exposure. Taken together, our data suggest a role for mitochondrial oxidative stress in CO toxicity at the cellular level during CO poisoning.

Acute iron poisoning in adult female

We report a case of a 27-year-old female with anemia, treated with high dose oral and parenteral iron therapy (within 20 days, the patient received a total dose of 4 g Fe+2 orally and 700 mg Fe+2 iv and im), and developed clinical manifestations characteristic of acute iron poisoning. Initial gastrointestinal symptoms and hypotension were followed by signs of mitochondrial toxicity: high leucocytosis, shock, multi-organ failure and disseminated intravascular coagulation. We discuss the difficulties in diagnosing acute iron poisoning. The initial low total iron blood capacity and high ferritin level, as well as the typical sequence of symptoms, supported the diagnosis. The patient avoided fatal consequences, probably due to the administration of iron doses over an extended period of time. However, cumulative effects led to the apparent iron toxicity. After 2 weeks of treatment, the patient was discharged from hospital in good condition. Human & Experimental Toxicology (2007) 26, 663-666.

A simplified acute physiology score in the prediction of acute organophosphate poisoning outcome in an intensive care unit

Organophosphate poisoning (OPP) occurs frequently and accounts for a large number of intoxication cases treated in intensive care units (ICU). Poisoning by these agents is a serious public health problem. Among pesticides, OPs are the main cause of poisoning and death in Loghman-Hakim Poison Center of Tehran, Iran. The aim of this study was to determine the impact of the Simplified Acute Physiology Score (SAPS II) in the prediction of mortality in patients with acute OPP requiring admission to the ICU of Loghman-Hakim Hospital Poison Center over a period of 12 months. This study was a prospective, case-control of records of patients with acute OPP admitted to the ICU between January 2006 and December 2006. The Demographic data were collected and SAPS II score was recorded. During the study period, 24 subjects were admitted to the ICU with acute OPP. All 24 patients (15 male) required endotracheal intubation and mechanical ventilation in addition to gastric decontamination and standard therapy with atropine and oximes and adequate hydration. Of these, 24 patients, eight (five male) died. SAPS II score was significantly higher in the non-survival group than the survival group. Mortality following acute OPP remains high despite adequate intensive care and specific therapy with atropine and oximes. One-third of the subjects needing intensive care die within the hospitalization period. SAPS II scores calculated within the first 24 hours were recognized as good prognostic indicator among patients with acute OPP that required ICU admission. It is concluded that SAPS II score above 11 within the first 24 hours is a predictor of poor outcome in patients with acute OPP requiring ICU admission.

Massive intoxication involving unusual high concentration of amitriptyline

Amitriptyline is a tricyclic antidepressant widely used in the treatment of depression. Antidepressant drugs are among the most commonly encountered causes of self-poisoning, as illustrated by several published cases in the literature. This investigation reports a case of massive amitriptyline intoxication, involving a 44-year old female found dead in bed. The presence of this tricyclic antidepressant was revealed by a routine screening procedure. The concentration was calculated by gas chromatography/ electron ionization-mass spectrometry in the selected ion monitoring mode after solid-phase extraction using proadifen as internal standard and was in the post-mortem whole blood sample 85.9 mug/mL. This value was much higher than the reported toxic values ever found in the literature, and may therefore have caused the victim's death. Nortriptyline was also detected in the toxic concentration range, as well as therapeutic levels of diazepam and nor-diazepam. Taking into account both the available circumstantial information and toxicological results, it is very likely that death was caused by self-poisoning. Human & Experimental Toxicology (2007) 26, 667-670.