Appropriate use of animal models in the assessment of risk during prenatal development: an illustration using inorganic arsenic

BACKGROUND: Assessing risks to human development from chemical exposure typically requires integrating findings from laboratory animal and human studies. METHODS: Using a case study approach, we present a program designed to assess the risk of the occurrence of malformations from inorganic arsenic exposure. We discuss how epidemiological data should be evaluated for quality and criteria for determining whether an association is causal. In this case study, adequate epidemiological data were not available for evaluating the potential effect of arsenic on development. Consequently, results from appropriately designed, conducted, and interpreted developmental toxicity studies, which have been shown to be predictive of human risk under numerous scenarios, were used. In our case study, the existing animal data were not designed appropriately to assess risk from environmental exposures, although such studies may be useful for hazard identification. Because the human and animal databases were deficient, a research program comprising modern guideline toxicological studies was designed and conducted. RESULTS: The results of those studies in rats, mice, and rabbits indicate that oral and inhalational exposures to inorganic arsenic do not cause structural malformations, and inhalational exposures produced no developmental effects at all. The new study results are discussed in conjunction with considerations of metabolism, toxicokinetics, and maternal toxicity. CONCLUSIONS: Based on the available experimental data, and absent contrary findings from adequately conducted epidemiological studies, we conclude that exposure to inorganic arsenic by environmentally relevant routes poses no risk of the occurrence of malformations and little risk of other prenatal developmental toxicity in developing humans without concomitant and near-lethal toxicological effects in mothers.     

The toxicity of 3-chloropropane-1,2-dipalmitate in Wistar rats and a metabonomics analysis of rat urine by ultra-performance liquid chromatography–mass spectrometry

3-Monochloropropane-1,2-diol(3-MCPD) fatty acid esters can release free 3-MCPD in a certain condition. Free 3-MCPD is a well-known food contaminant and is toxicological well characterized, however, in contrast to free 3-MCPD, the toxicological characterization of 3-MCPD fatty acid esters is puzzling. In this study, toxicological and metabonomics studies of 3-chloropropane-1,2-dipalmitate(3-MCPD dipalmitate) were carried out based on an acute oral toxicity test, a 90-day feeding test and ultra-performance liquid chromatography–mass spectrometry (UPLC–MS) analysis. The LD₅₀ value of 3-MCPD dipalmitate was determined to be 1780 mg/kg body weight (bw) for Wistar rats. The results of the 90-day feeding test in male Wistar rats showed that 3-MCPD dipalmitate caused a significant increase in blood urea nitrogen and creatinine in the high-dose group (267 mg/kg bw/day) compared to control rats. Renal tubular epithelium cell degeneration and renal tubular hyaline cast accumulation were the major histopathological changes in rats administered 3-MCPD dipalmitate. Urine samples obtained after the 90-day feeding test and analyzed by UPLC–MS showed that the differences in metabolic profiles between control and treated rats were clearly distinguished by partial least squares-discriminant analysis (PLS-DA) of the chromatographic data. Five metabolite biomarkers which had earlier and significant variations had been identified, they were first considered to be the early, sensitive biomarkers in evaluating the effect of 3-MCPD dipalmitate exposure, and the possible mechanism of these biomarkers variation was elucidated. The combination of histopathological examination, clinical chemistry and metabolomics analyses in rats resulted in a systematic and comprehensive assessment of the long-term toxicity of 3-MCPD dipalmitate.     

The effects of repeated oral gavage on the health of male CD-1 mice

Oral gavage is a widely used method for administering substances to animals in pharmacological and toxicological studies. The authors evaluated whether oral gavage causes behavioral indicators of stress, increased mortality rate, alterations in food and water consumption and body weight or histological lesions in CD-1 mice. Gavage was carried out once per d for 5 d per week over 6 consecutive weeks. The mortality rate of mice in this study was 15%. Mice subjected to gavage did not undergo changes in food or water consumption during the study, and their mean body weights and relative organ weights were similar to those of mice in the control group. Serum cortisol levels at the time of euthanasia in mice in both groups were within the normal range. Histopathology showed acute esophagitis and pleurisy, indicative of perforation of the esophagus, in the two mice that died but no abnormalities in the other mice. The results suggest that animal stress and mortality related to oral gavage can be minimized when the procedure is carried out by an experienced technician.     

Hematological,biochemical, respiratory,cardiovascular and electroneurophysiological parameters in African green monkeys (Cercopithecus aethiops sabaeus). Its use in non‐clinical toxicological studies

Background The purpose of this study is to better characterize the hematological, biochemical, respiratory, cardiovascular and electroneurophysiological parameters in young adult Cercopithecus aethiops sabaeus of both sexes. The rhesus and cynomolgus monkeys are widely used as experimental primate models. However, only few articles have been published testing toxicological effects of pharmaceuticals on African green monkey. Methods The present study was carried out with the recompilation of all parameters recorded before the first drug administration in five sub‐chronic or chronic toxicological studies performed on 66 Cercopithecus aethiops sabaeus, born in Cuba. Results This study provides hematological, biochemical, respiratory, cardiovascular and electroneurophysiological data for both choosing animals to be included into experiments and monitoring these parameters during the study. Conclusions We conclude that this study provides valuable integrated data for determining the health status, including electroneurophysiological parameters, data not previously reported for this species, of the African green monkey.     

Biodistribution and Clearance of TiO2 Nanoparticles in Rats after Intravenous Injection

Titanium dioxide (TiO₂) nanoparticles are used in many applications. Due to their small size, easy body penetration and toxicological adverse effects have been suspected. Numerous studies have tried to characterize TiO₂ translocation after oral, dermal or respiratory exposure. In this study, we focused on TiO₂ nanoparticle biodistribution, clearance and toxicological effects after intravenous injection, considering TiO₂ translocation in the blood occurs. Using ICP-OES, transmission electron microscopy, and histological methods, we found TiO₂ accumulation in liver, lungs and spleen. We estimated TiO₂ nanoparticles’ half life in the body to about 10 days. Clinical biomarkers were also quantified for 56 days to identify potential toxicological impact on lungs, blood, liver, spleen and kidneys. Results showed absence of toxicological effects after TiO₂ intravenous injection at concentrations of 7.7 to 9.4 mg/kg.     

Establishing Causality between Exposure to Metals and Effects on Fish

This study evaluates causal relationships between chronic exposure of fish to metals and effects at different levels of biological organization based on a weight-of-evidence approach. Criteria for evaluation of causality were strength, consistency, and specificity of the association, as well as biological gradient and plausibility. Field sampling was conducted three times between 1998 and 2000, in Furnas Stream, impacted by an abandoned lead mine, and in three other locations, including two reference and one impacted sites. Levels of Pb, Zn, Cd, and Ag in sediments from the Furnas Stream exceeded background levels, and their concentrations were above sediment quality guidelines. Residual levels of metals in fish tissue were high enough to indicate reduced growth, reproduction and/or survival according to toxicological benchmarks. Lead-induced biochemical changes (ALA-D activity depletion) were observed in two species of siluriform catfish. The condition factor of a predatory catfish was reduced, and the percentage of prey generalists was higher in Furnas than at the noncontaminated sites. Reduction in fish community diversity and density was observed. Integration of data provided supporting evidence that observed effects on fish from the Furnas Stream resulted from long-term exposure to metals, however influences from other stressors cannot be ruled out.     

Determination of atrazine and its metabolites in mouse urine and plasma by LC-MS analysis

Atrazine is a herbicide widely used on agricultural commodities. Existing analytical methods to analyze atrazine and its metabolites in biological matrices have various drawbacks. Thus, further development of such methods will be needed to correlate the growing number of toxicological effects associated with atrazine exposure with the concentrations of this compound and its metabolites in plasma, urine, and tissues. The purpose of this study was to develop a broad and sensitive LC-MS method for the analysis of atrazine and its metabolites in mouse urine and plasma. We were able to simultaneously measure atrazine and its major mammalian metabolites, which include didealkyl atrazine, desisopropyl atrazine, desethyl atrazine, atrazine-glutathione conjugate, and atrazine-mercapturate, using preparation procedures that used small sample volumes of plasma and urine (0.25 and 0.5 ml, respectively). Furthermore, derivatization of analytes prior to analysis was unnecessary. This method was used to analyze plasma and urine samples following single in vivo oral exposures of a limited number of mice to atrazine (doses, 5-250 mg/kg body weight) to demonstrate the utility of this LC-MS method. The data obtained from this study suggest that atrazine is rapidly metabolized in mice. Didealkyl atrazine was the most abundant metabolite detected in the urine and plasma samples (approximately 1000 microM in 24-h urine and approximately 100 microM in plasma following the highest dose of atrazine), with lesser quantities of mono N-dealkylated metabolites and thio conjugates of atrazine observed. We also used this methodology in a preliminary study of cytochrome P450-catalyzed metabolism of atrazine in vitro. The results obtained in this study suggest that this method will be a useful tool for the determination of atrazine and its metabolites in future pharmacokinetic studies and for the subsequent development and refinement of biologically based models of atrazine disposition.     

Fluoride Ingestion After Brushing with a Gel Containing a High Concentration of Fluoride

The aim of the study was to evaluate the amount of fluoride remaining in the oral cavity of children after brushing with fluoride gel (1.25% F). The study involved six groups of 7-year-old and six groups of 11-year-old children. The procedure was carried out according to the manufacturer’s recommendations. Fluoride concentrations were determined using ion-selective fluoride electrode. No statistically significant difference was found between the amount of fluorides that remained in the oral cavity of younger and older age group (1.2 and 1.3 mg, respectively; p > 0.05). The amount of fluorides swallowed during the procedure in both age groups proves to be within acceptable limit, as far as risk of acute poisoning symptoms is concerned. The individual daily fluoride exposure during the day of procedure seems to be twice as high compared to average fluoride intake from diet and dentifrice, and it does not exceed Tolerable Upper Intake Level for children more than 8. In younger children, it seems justifiable to reduce the amount of the preparation applied on a toothbrush, especially when daily use of the gel is recommended.     

Tolerance and safety of vitamin E: a toxicological position report.

From numerous publications on the "prophylactic" and "therapeutic" use of vitamin E, it may be concluded that the toxicity of vitamin E is very low. It has been demonstrated in animal experiments that vitamin E has neither mutagenic, teratogenic nor carcinogenic properties. Based on studies in humans, a daily dosage of 100-300 mg vitamin E can be considered harmless from a toxicological point of view. Using double-blind studies involving a large number of subjects, it has been demonstrated that large oral doses of up to 3,200 USP-Units/day led to no consistent adverse effects. From a large body of published data, dosage ranges have been deduced which can be characterized as safe for human subjects even where their use extends over a long period of time. It should, however, be noted that oral intake of high levels of vitamin E can exacerbate the blood coagulation defect of vitamin K deficiency caused by malabsorption or anticoagulant therapy. High levels of vitamin E intake are, therefore, contraindicated in these subjects.     

Derivation of Environmental Soil Quality Guidelines for 2,4,6-Trinitrotoluene Using the CCME Approach

The Protocol for the Derivation of Environmental and Human Health Soil Quality Guidelines of the Canadian Council of Ministers of the Environment was used to prepare preliminary Environmental Soil Quality Guidelines (SQG_E) for 2,4,6-trinitrotoluene (TNT). A thorough literature search led to the compilation of the existing toxicological data. Calculated Environmental Soil Quality Guidelines for TNT are SQG_E=0.02?mg/kg (preliminary value) for agricultural land use and 86?mg/kg for residential/park, commercial/industrial land use. Because of the lack of sufficient scientific information on the effects of TNT in soil on microbial processes, this type of evaluation was not possible. For oral toxicological data, laboratory animals were used instead of grazing and foraging species to determine the ingestion guideline since no related literature was found. Furthermore, this work has led to the identification of avenues of future research necessary to complete the task at hand. The research should focus on specific studies involving direct contact between the organisms and the soil, in order to: (1) develop a database of effects of TNT on microbial processes, (2) study the effects of TNT ingestion on birds and grazing herbivores, (3) determine plant bioconcentration factors, and (4) observe in situ the toxic effects after direct contact exposure.     

Relevance of clonogenic assays in hematotoxicology

Clonogenic assays have been established in hematology for 30 years. They have been widely used in fundamental studies on hematopoiesis and they are also routinely used in clinical hematology to confirm diagnosis or to predict time to recovery in cases of bone marrow failure. Their use in toxicological studies is more recent. Adverse effects of xenobiotics can induce hematological problems and pathologies such as neutropenia, thrombocytopenia, anemia, and aplastic anemia. Three clonogenic assays are proposed for granulopoiesis, megakaryopoieisis and erythropoieisis. Hematopoietic progenitors from murine or human origin can be cultured in the presence of xenobiotics using validated protocols to complete standard animal toxicological studies. These clonogenic assays can help to predict adverse effects of drugs or toxicants. Clonogenic assays using white blood cell progenitors (CFU-GM culture) have recently been validated by ECVAM and can be used routinely. Megakaryocyte progenitor (CFU-MK) culture is under development and prevalidation in toxicological studies supported by ECVAM. Red blood cells progenitor culture (BFU-E) has been proposed but needs international validation to be recognized. This revised version was published online in July 2006 with corrections to the Cover Date.     

A path forward for improved noncancer assessment: the truly adverse dose (the TAD)

Conventional noncancer assessment involves the computation of hazard quotients (HQ), simple ratios of an individual’s or a population’s estimated chemical intake, and a reference dose (the RfD) that is held to reflect a safe level of exposure. HQs?>?1, indicating RfD exceedance, have considerable uncertainty for two reasons. It is not known that the critical effect of the animal study that supports the RfD is adverse (i.e., harmful), and it is unclear at what dose higher than an RfD, truly health compromising effects are triggered. Through methodically reviewing critical studies of the U.S. EPA’s IRIS database, we investigated the efficacy of the present assessment scheme with its considerable emphasis on RfD development. Aside from noted inconsistencies in reporting, and a substantial percentage of oral noncarcinogens having a less than ideal toxicological review source, our analysis found numerous instances of absent toxicological information to fuel HQ computation, with this largely a function of the dated nature of the supporting studies. In light of our analysis, we propose a recommended replacement scheme for noncancer assessment. In place of estimating the degree to which RfDs are exceeded, we recommend determining whether Truly Adverse Doses (TADs), to be newly developed, are approached.     

Long term health effects of low dose exposure to nerve agent

Possible long-term toxic effcts of nerve agents have been investigated using sensitive toxicological screens and extensive toxicity studies in various animal models. Data on humans have been obtained from controlled studies and accidental exposures. Studies in the area of ‘low dose’ exposure to nerve agents are currently being performed.     

Effect of 900 MHz radio frequency radiation on beta amyloid protein, protein carbonyl, and malondialdehyde in the brain

Recently, many studies have been carried out in relation to 900 MHz radiofrequency radiation (RF) emitted from a mobile phone on the brain. However, there is little data concerning possible mechanisms between long-term exposure of RF radiation and biomolecules in brain. Therefore, we aimed to investigate long-term effects of 900 MHz radiofrequency radiation on beta amyloid protein, protein carbonyl, and malondialdehyde in the rat brain. The study was carried out on 17 Wistar Albino adult male rats. The rat heads in a carousel were exposed to 900 MHz radiofrequency radiation emitted from a generator, simulating mobile phones. For the study group (n: 10), rats were exposed to the radiation 2 h per day (7 days a week) for 10 months. For the sham group (n: 7), rats were placed into the carousel and the same procedure was applied except that the generator was turned off. In this study, rats were euthanized after 10 months of exposure and their brains were removed. Beta amyloid protein, protein carbonyl, and malondialdehyde levels were found to be higher in the brain of rats exposed to 900 MHz radiofrequency radiation. However, only the increase of protein carbonyl in the brain of rats exposed to 900 MHz radiofrequency radiation was found to be statistically significant (p<0.001). In conclusion, 900 MHz radiation emitted from mobile/cellular phones can be an agent to alter some biomolecules such as protein. However, further studies are necessary.     

Automated weighing procedure for toxicological studies on small animals, using a minicomputer.

A compact system was designed for weighing procedures in toxicological studies on small animals that integrated 4 basic functions: data acquistion, record keeping, statistical analysis, and report preparation. An electric balance, a minicomputer, and a typewriter were incorporated into the system. Elimination of clerical work and accelerated flow of information between planning, operation, and evaluation of experiments were found to be the main advantages.     

Gold and palladium burden from dental restoration materials.

From 81 volunteers (16 without dental restorations, 65 with gold crowns or inlays) samples of saliva before and after chewing gum, blood, serum, urine and faeces were taken and analysed for gold (Au) and palladium (Pd). The Au concentration in all analysed biomonitors correlates significantly to the number of teeth with gold restorations. For Pd the correlations were still significant, but weaker than for Au. Persons with gold restorations show maximal Au and Pd concentrations, 10(2)-10(3) higher than the background burden. The calculated maximal daily Au load in saliva (1.38 mg Au per day) reaches the range of an oral Au therapy for rheumatoid arthritis with 6 mg Auranofin (= 1.74 mg Au per day). During this therapy severe and frequent side effects are reported. In contrast, the Au concentration in serum maximally reached from Au restorations, amounts to only approximately 1/20 of the Au level during arthritis therapy. But even under subtherapeutic doses of 1 mg Auranofin/day severe side effects have been reported (4 out of 56 cases). The mean Au blood concentration from 1 mg Auranofin daily was only 3 times higher than our maximum value. A toxicological classification of the Pd values is difficult, because no toxicological threshold limit has been established, especially for the low-level long-term burden with Pd.     

Microbiological and toxicological quality of dried herbs

Aims: The microbiological and toxicological quality of 51 samples of dried herbs (Melissa officinalis, Salvia officinalis, Malva sylvestris, Matricaria chamomilla, Alchemilla vulgaris and Centaurea cyanus) cultivated in family‐managed farms in Molise Region (Italy) was evaluated. Methods and Results: All the samples were analysed by using conventional methods, and for samples preparation, an alternative Washing and Shaking (WaS) protocol was developed to reduce release of antimicrobial compounds. None of the samples were of unsatisfactory quality with respect to aflatoxin B1, and only three samples from Malva sylvestris exceeded the limit of total aflatoxins according to Recommendation 2004/24/EC. The International Commission on Microbiological Specifications for Foods limits for mesophilic bacteria and total coliforms were exceeded in the 29·4 and 3·9% of samples, respectively: 7·8% of samples also exceeded the limit for Escherichia coli established by European Spice Association. When the ‘WaS’ method was used, higher microbial counts were obtained, especially for A. vulgaris, S. officinalis and M. officinalis. Conclusions: Herbs cultivated in family‐managed small agricultural areas showed a good microbiological and toxicological quality, irrespectively of preliminary washing or selection procedures. Significance and Impact of the Study: Herb matrices may contain antimicrobial activity which should be considered when applying the conventional microbiological methods for sample preparation. Alternative preparation protocols may have advantages to reduce antimicrobial effects and should be further evaluated.     

气候变化对鸟类影响：长期研究的意义

过去一个多世纪全球气候发生了明显变化,地球表面温度正在逐渐变暖。已有大量研究结果表明,鸟类已经在种群动态变化、生活史特性以及地理分布范围等方面对全球气候变化作出了相应的反应。根据全球范围内气候变化对鸟类影响的研究资料,尤其是北美和欧洲的一些长期研究项目的成果,综述了气候变化对鸟类分布范围、物候、繁殖和种群动态变化等方面的可能影响。这些长期研究项目为探讨气候变化在个体和种群的水平上如何长时间地影响鸟类提供了独特的机会,对未来中国鸟类学研究也会有所裨益。     

NAT2 polymorphisms with oral carcinoma susceptibility: a meta-analysis

Published data have implicated NAT2 polymorphisms as risk factors for various cancers. A number of studies have focused on the association of NAT2 polymorphisms with susceptibility to oral carcinoma and have yielded inconclusive results. The aim of the present study was to derive a more precise estimation of the relationship. We first carried out a deliberate search in the databases without a language limitation, covering all papers published up to Dec 2011. A total of seven case-control studies including 1,379 cases and 1,868 controls were selected and the relevant data were extracted for systematic meta-analyses. No significant association was found for the overall data (OR: 1.04, 95?% CI: 0.79-1.39). In subgroup analyses according to ethnicity, slow acetylators might increase oral cancer risk among Asians (OR: 1.38, 95?% CI: 1.04-1.82) but not Caucasians or Mixed races. The data suggested that NAT2 polymorphisms might be a low-penetrant risk factor for oral carcinoma in Asians.